Heterotandem bicyclic peptide complexes

ABSTRACT

The present invention relates to heterotandem bicyclic peptide complexes which comprise a first peptide ligand, which binds to a component present on a cancer cell, conjugated via a linker to a second peptide ligand, which binds to a component present on an immune cell. The invention also relates to the use of said heterotandem bicyclic peptide complexes in preventing, suppressing or treating cancer.

FIELD OF THE INVENTION

The present invention relates to heterotandem bicyclic peptide complexeswhich comprise a first peptide ligand, which binds to a componentpresent on a cancer cell, conjugated via a linker to a second peptideligand, which binds to a component present on an immune cell. Theinvention also relates to the use of said heterotandem bicyclic peptidecomplexes in preventing, suppressing or treating cancer.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Dec. 9, 2020, and named 176531_SL_20201209. txt(35,338 bytes), the contents of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and targetspecificity to protein targets and hence are an attractive moleculeclass for the development of therapeutics. In fact, several cyclicpeptides are already successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008),Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result froma relatively large interaction surface formed between the peptide andthe target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), acyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclicpeptide inhibitor upain-1 binding to urokinase-type plasminogenactivator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8 (MMP-8) which lost its selectivity over other MMPswhen its ring was opened (Cherney et al. (1998), J Med Chem 41 (11),1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin andactinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp and McNamara(1985), J. Org. Chem; Timmerman et al.

(2005), ChemBioChem). Meloen and co-workers had usedtris(bromomethyl)benzene and related molecules for rapid andquantitative cyclisation of multiple peptide loops onto syntheticscaffolds for structural mimicry of protein surfaces (Timmerman et al.(2005), ChemBioChem). Methods for the generation of candidate drugcompounds wherein said compounds are generated by linking cysteinecontaining polypeptides to a molecular scaffold as for exampletris(bromomethyl)benzene are disclosed in WO 2004/077062 and WO2006/078161.

Phage display-based combinatorial approaches have been developed togenerate and screen large libraries of bicyclic peptides to targets ofinterest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009/098450). Briefly, combinatorial libraries of linear peptidescontaining three cysteine residues and two regions of six random aminoacids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclisedby covalently linking the cysteine side chains to a small molecule(tris-(bromomethyl)benzene).

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided aheterotandem bicyclic peptide complex comprising:

-   -   (a) a first peptide ligand which binds to a component present on        a cancer cell; conjugated via a linker to    -   (b) a second peptide ligand which binds to a component present        on an immune cell;        wherein each of said peptide ligands comprise a polypeptide        comprising at least three reactive groups, separated by at least        two loop sequences, and a molecular scaffold which forms        covalent bonds with the reactive groups of the polypeptide such        that at least two polypeptide loops are formed on the molecular        scaffold, characterised in that said heterotandem bicyclic        peptide complex comprises the following first and second peptide        ligands:

Heterotandem Complex No. First Peptide Second Peptide BCY12229[Ac]D[HArg]CSAGWLTMCQKLHLCPSHAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 1; BCY11865)ID NO: 67; BCY8928) BCY12230 [Ac]D[HArg]CSKGWLTMCQK(Ac)LHLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 2; BCY11866)ID NO: 67; BCY8928) BCY12231 [Ac]D[HArg]CSAGWLTKCQK(Ac)LHLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 3; BCY11867)ID NO: 67; BCY8928) BCY12232 [Ac]D[HArg]CSAGWLTMCKK(Ac)LHLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 4; BCY11868)ID NO: 67; BCY8928) BCY12242 [Ac]D[HArg]CSAGWLTMCQK(Ac)LKLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 5; BCY11869)ID NO: 67; BCY8928) BCY12375 Ac-SDKCSAGWLTMCQK[PYA]LHLCPSH (SEQ ID NO:[Ac]C[tBuAla]EE(dK)PYCFADPY[Nle]C[Dap(PYA)] 6; BCY10861)(SEQ ID NO: 68; BCY12023) BCY12663[Ac]SD[HArg]CSAGWLTMCQ[HArg]LHLCPSHK (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 7; BCY12479)ID NO: 67; BCY8928) BCY12796 [Ac]SD[HArg]CSAGWLTMC[HArg]QLNLCPSHK (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 8; BCY12477)ID NO: 67; BCY8928) BCY12021 Ac-SDKCSAGWLTMCQK[PYA]LHLCPSH (SEQ ID NO:[Ac]C[tBuAla]PE[dK]PYCFADPY[Nle]C[Dap(PYA)]  9; BCY10861)(SEQ ID NO: 69; BCY11144) BCY12233[PYA]A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 10; BCY11813)NO: 70; BCY8920) BCY12234 [Ac]A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CK(PYA)Ac-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 11; BCY11814)NO: 70; BCY8920) BCY12235 [Ac]A[HArg]DC[HyP]LVNPLCLK(PYA)P[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 12; BCY11815)NO: 70; BCY8920) BCY12236 [Ac]A[HArg]DC[HyP]K(PYA)VNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 13; BCY11816)NO: 70; BCY8920) BCY12237 [Ac]A[HArg]DC[HyP]LVNPLCK(PYA)HP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 14; BCY11817)NO: 70; BCY8920) BCY12711 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]EE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 71; BCY12143) BCY12712 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 72; BCY12149) BCY12713 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFANPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 73; BCY12147) BCY12714 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFAEPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 74; BCY12145) BCY12715 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFA[Aad]PY[Nle]C NO: 15; BCY9594)(SEQ ID NO: 75; BCY12146) BCY12717A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle] NO: 15; BCY9594)[Cysteamine] (SEQ ID NO: 76; BCY12352) BCY12718A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID [3-mercaptopropionicNO: 15; BCY9594) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ IDNO: 77; BCY12353) BCY12719 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[3-mercaptopropionic NO: 15; BCY9594)acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle][Cysteamine] (SEQ ID NO: 78; BCY12354) BCY12720A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID Palmitic acid-yGly-yGlu-NO: 15; BCY9594) C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO:79; BCY12360) BCY12961 [Ac]A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CKAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 16; BCY12734)ID NO: 67; BCY8928) BCY12962[Ac]A[HArg]DC[HyP]LVNPLCLKP[dD]W[HArg]C (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 17; BCY12735)ID NO: 67; BCY8928) BCY12963[Ac]A[HArg]DC[HyP]KVNPLCLHP[dD]W[HArg]C (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 18; BCY12736)ID NO: 67; BCY8928) BCY12964[Ac]A[HArg]DC[HyP]LVNPLCKHP[dD]W[HArg]C (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 19; BCY12737)ID NO: 67; BCY8928) BCY12965 A[HArg]DC[HyP]LVNPLCLHP[dE]V[HArg]C (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 20; BCY12738)ID NO: 67; BCY8928) BCY12966 A[HArg]EC[HyP]LVNPLCLHP[dE]V[HArg]C (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 21; BCY12739)ID NO: 67; BCY8928) BCY13029 A[HArg]DC[HyP]LVNPLCLEP[dD]W[HArg]C (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 22; BCY12854)ID NO: 67; BCY8928) BCY13030 A[HArg]DC[HyP]LVNPLCLHP[dD]WTC (SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 23; BCY12855)ID NO: 67; BCY8928) BCY13031 A[HArg]DC[HyP]LVNPLCLEP[dD]WTC (SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 24; BCY12856)ID NO: 67; BCY8928) BCY13032 A[HArg]DC[HyP]LVNPLCLEP[dD]WTC[dA] (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 25; BCY12857)ID NO: 67; BCY8928) BCY13033 A[HArg]DC[HyP]LVNPLCLEP[dA]WTC (SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 26; BCY12858)ID NO: 67; BCY8928) BCY13034 A[HArg]DC[HyP]LVNPLCL[33DPA]P[dD]WTC (SEQ Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 27; BCY12859)ID NO: 67; BCY8928) BCY13035 C[HyP]LVNPLCL[33DPA]P[dD]WTC SEQ ID NO: 28;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY12860)ID NO: 67; BCY8928) BCY13036 C[HyP]LVNPLCLEP[dD]WTC[dA] (SEQ ID NO: 29;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY12861)ID NO: 67; BCY8928) BCY13037 A[HArg]DC[HyP][Cba]VNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 30; BCY12862)ID NO: 67; BCY8928) BCY13038 A[HArg]DC[HyP][Cba]VNPLCLEP[dD]WTC (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 31; BCY12863)ID NO: 67; BCY8928) BCY13039 [dA][HArg]DC[HyP][Cba]VNPLCLEP[dD]WTC[dA]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 32; BCY12864)ID NO: 67; BCY8928) BCY13040 C[HyP][Cba]VNPLCL[33DPA]P[dD]WTC[dA]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 33; BCY12865)(SEQ ID NO: 67; BCY8928) BCY13041A[HArg]DC[HyP]LVNPLCL[33DPA]P[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 34; BCY12866)ID NO: 67; BCY8928) BCY13141 A[HArg]DC[HyP]LVNPLCLEP[dD]WTC (SEQ ID NO:[3-mercaptopropionic 24; BCY12856)acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 77; BCY12353)BCY13142 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[3-mercaptopropionic NO: 15; BCY9594) acid][tBuAla]EE[dK]PYCFADPY[Nle]C(SEQ ID NO: 80; BCY13137) BCY13143A[HArg]DC[HyP]LVNPLCLEP[dD]WTC (SEQ ID NO: [3-mercaptopropionic24; BCY12856) acid][tBuAla]EE[dK]PYCFADPY[Nle]C(SEQ ID NO: 80; BCY13137) BCY13250A[HArg]DC[HyP]LVNPLCLHP[d1Nal]W[HArg]C (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 35; BCY13116)ID NO: 67; BCY8928) BCY13251 A[HArg]DC[HyP]LVNPLCL[1Nal]P[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 36; BCY13117)ID NO: 67; BCY8928) BCY13252 A[HArg]DC[HyP]LVNPLCLEP[d1Nal]WTCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 37; BCY13118)(SEQ ID NO: 67; BCY8928) BCY13253C[HyP]LVNPLCLONalp[dD]WTC (SEQ ID NO: 38;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA BCY13119)(SEQ ID NO: 67; BCY8928) BCY13254 [Ac]C[HyP]LVNPLCL[33DPA]P[dD]WTC[dK]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 39; BCY13120)(SEQ ID NO: 67; BCY8928) BCY13255[NMeAla][HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 40; BCY13121)ID NO: 67; BCY8928) BCY13256 [NMeAla][HArg]DC[HyP]LVNPLCLEP[dD]WTC (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 41; BCY13122)ID NO: 67; BCY8928) BCY13257 [dA][HArg]DC[HyP][Cba]VNPLCLEP[dA]WTC[dA]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 42; BCY13123)ID NO: 67; BCY8928) BCY13258[d1Nal][HArg]DC[HyP][Cba]VNPLCLEP[dA]WTC[dA]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 43; BCY13124)ID NO: 67; BCY8928) BCY13260 [dA]EDC[HyP]LVNPLCLEP[dD]WTC SEQ ID NO: 44;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY13126)ID NO: 67; BCY8928) BCY13261 [dA][dA]DC[HyP]LVNPLCLEP[dD]WTC SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 45; BCY13127)ID NO: 67; BCY8928) BCY13262 ADC[HyP]LVNPLCLEP[dD]WTC (SEQ ID NO: 46;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY13128)ID NO: 67; BCY8928) BCY13264 A[HArg]DC[HyP][hGlu]VNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 47; BCY13130)ID NO: 67; BCY8928) BCY13265 A[HArg]DC[HyP]LVNPLC[hGlu]HP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 48; BCY13131)ID NO: 67; BCY8928) BCY13266 A[HArg]DC[HyP]LVNPLCL[hGlu]P[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 49; BCY13132)ID NO: 67; BCY8928) BCY13268 A[HArg]DC[HyP]LVNPLCLHP[dNle]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 50; BCY13134)ID NO: 67; BCY8928) BCY13269A[HArg]DC[HyP]LVNPLCL[Nle]P[dD]W[HArg]C (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 51; BCY13135)ID NO: 67; BCY8928) BCY13340C[HyP][Cba]VNPLCL[33DPA]P[dD]WTC[dA] (SEQ ID [3-mercaptopropionicNO: 33; BCY12865) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ IDNO: 77; BCY12353) BCY13342 C[HyP]LVNPLCL[33DPA]P[dD]WTC (SEQ ID NO: 28;[3-mercaptopropionic BCY12860)acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 77; BCY12353)BCY11616 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:Ac-ACIEE(D-K)(PYA)QYCFADPY(Nle)CA (SEQ ID NO: 52; BCY8116) 81; BCY7744)BCY12238 [Ac]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WC (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 53; BCY12024)ID NO: 67; BCY8928) BCY12377 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[Ac]C[tBuAla]EE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID 52; BCY8116)NO: 71; BCY12143) BCY12379 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID 52; BCY8116)NO: 72; BCY12149) BCY12572 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle] 52; BCY8116)[Cysteamine] (SEQ ID NO: 76; BCY12352) BCY12573CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO: [3-mercaptopropionic52; BCY8116) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ IDNO: 77; BCY12353) BCY12574 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[3-mercaptopropionic 52; BCY8116) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle][Cysteamine] (SEQ ID NO: 78; BCY12354) BCY12575CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO: Palmitic acid-yGly-yGlu-52; BCY8116) C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO:79; BCY12360) BCY12576 [3-mercaptopropionicAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQacid]P[1Nal][dK]CM[HArg]DWSTP[HyP]WC (SEQ ID ID NO: 67; BCY8928)NO: 54; BCY12363) BCY12577 [Ac]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ[Cysteamine] (SEQ ID NO: 55; BCY12364) ID NO: 67; BCY8928) BCY12578[3-mercaptopropionic Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQacid]P[1Nal][dK]CM [HArg]DWSTP[HyP]W ID NO: 67; BCY8928)[Cysteamine] (SEQ ID NO: 56; BCY12365) BCY12579[Ac]CP[1Nal][dK]CM[HArg]HWSTP[HyP]WC (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 57; BCY12366)ID NO: 67; BCY8928) BCY12580[Ac]CP[1Nal][dK]CM[HArg]EWSTP[HyP]WC (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 58; BCY12367)ID NO: 67; BCY8928) BCY12581 CP[1Nal][dE]CM[HArg]DWSTP[HyP]WC SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 59; BCY12368)ID NO: 67; BCY8928) BCY12582 CP[1Nal][dA]CM[HArg]DWSTP[HyP]WC SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 60; BCY12369)ID NO: 67; BCY8928) BCY12583 CP[1Nal][dE]CL[HArg]DWSTP[HyP]WC SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 61; BCY12370)ID NO: 67; BCY8928) BCY12584 Palmitic-yGlu-yGlu-Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQCP[1Nal][dK]CM[HArg]DWSTP[HyP]WC SEQ ID NO: ID NO: 67; BCY8928)62; BCY12371) BCY12585 CP[1Nal][dE]CM[HArg]EWSTP[HyP]WC SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 63; BCY12384)ID NO: 67; BCY8928) BCY12709 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFAD[NMeAla]Y[Nle]C 52; BCY8116)(SEQ ID NO: 82; BCY12381) BCY12710CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFAD[NMeDAla]Y[Nle]C 52; BCY8116)(SEQ ID NO: 83; BCY12382) BCY11468[PYA][B-Ala]CP[1Nal][dD]CM[HArg]DWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 64; BCY11016)ID NO: 67; BCY8928) BCY11618[PYA][B-Ala]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 65; BCY11143)NO: 70; BCY8920) BCY11776 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[Ac]C[tBuAla]PE[dK]PYCFADPY[Nle]C[Dap(PYA)] 52; BCY8116)(SEQ ID NO: 69; BCY11144) BCY11860[PYA][B-Ala]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 65; BCY11143)NO: 70; BCY8920) BCY12020 [PYA][B-Ala]CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC[Ac]C[tBuAla]PE[dK]PYCFADPY[Nle]C[Dap(PYA)] (SEQ ID NO: 64; BCY11016)(SEQ ID NO: 69; BCY11144) BCY12661 [PYA]CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC[Ac]C[tBuAla]EE(dK)PYCFADPY[Nle]C[Dap(PYA)] (SEQ ID NO: 66; BCY11015)(SEQ ID NO: 68; BCY12023) BCY12969CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C[1,2- 52; BCY8116)diaminoethane] (SEQ ID NO: 84; BCY12358)wherein 1Nal represents 1-naphthylalanine, HArg represents homoarginine,HyP represents hydroxyproline, B-Ala represents beta-alanine, PYArepresents 4-pentynoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cbarepresents β-cyclobutylalanine, hGlu represents homoglutamic acid, Nlerepresents norleucine, NMeAla represents N-methyl-alanine, tBuAlarepresents t-butyl-alanine, Aad represents alpha-L-aminoadipic acid, Acrepresents an acetyl group, Dap represents diaminopropionic acid, or apharmaceutically acceptable salt thereof.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a heterotandem bicyclic peptidecomplex as defined herein in combination with one or morepharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided aheterotandem bicyclic peptide complex as defined herein for use inpreventing, suppressing or treating cancer.

DETAILED DESCRIPTION OF THE INVENTION

First Peptide Ligands

References herein to the term “cancer cell” includes any cell which isknown to be involved in cancer. Cancer cells are created when the genesresponsible for regulating cell division are damaged. Carcinogenesis iscaused by mutation and epimutation of the genetic material of normalcells, which upsets the normal balance between proliferation and celldeath. This results in uncontrolled cell division and the evolution ofthose cells by natural selection in the body. The uncontrolled and oftenrapid proliferation of cells can lead to benign or malignant tumours(cancer). Benign tumors do not spread to other parts of the body orinvade other tissues. Malignant tumors can invade other organs, spreadto distant iodations (metastasis) and become life-threatening.

In one embodiment, the cancer cell is selected from an HT1080, A549,SC-OV-3, PC3, H1376, NCI-H292, LnCap, MC38, 4T1-D02 and RKO tumor cell.

In one embodiment, the component present on a cancer cell is Nectin-4.

Nectin-4 is a surface molecule that belongs to the nectin family ofproteins, which comprises 4 members. Nectins are cell adhesion moleculesthat play a key role in various biological processes such as polarity,proliferation, differentiation and migration, for epithelial,endothelial, immune and neuronal cells, during development and adultlife. They are involved in several pathological processes in humans.They are the main receptors for poliovirus, herpes simplex virus andmeasles virus. Mutations in the genes encoding Nectin-1 (PVRL1) orNectin-4 (PVRL4) cause ectodermal dysplasia syndromes associated withother abnormalities. Nectin-4 is expressed during foetal development. Inadult tissues its expression is more restricted than that of othermembers of the family. Nectin-4 is a tumour-associated antigen in 50%,49% and 86% of breast, ovarian and lung carcinomas, respectively, mostlyon tumours of bad prognosis. Its expression is not detected in thecorresponding normal tissues. In breast tumours, Nectin-4 is expressedmainly in triple-negative and ERBB2+ carcinomas. In the serum ofpatients with these cancers, the detection of soluble forms of Nectin-4is associated with a poor prognosis. Levels of serum Nectin-4 increaseduring metastatic progression and decrease after treatment. Theseresults suggest that Nectin-4 could be a reliable target for thetreatment of cancer. Accordingly, several anti-Nectin-4 antibodies havebeen described in the prior art. In particular, Enfortumab Vedotin(ASG-22ME) is an antibody-drug conjugate (ADC) targeting Nectin-4 and iscurrently clinically investigated for the treatment of patientssuffering from solid tumours.

In one embodiment, the first peptide ligand comprises a Nectin-4 bindingbicyclic peptide ligand.

Suitable examples of Nectin-4 binding bicyclic peptide ligands aredisclosed in PCT Patent Application No PCT/GB2019/051740, the peptidesof which are incorporated herein by reference.

In one embodiment, the Nectin-4 binding bicyclic peptide is selectedfrom any of the peptides of SEQ ID NOS: 52 to 66 described herein.

In an alternative embodiment, the component present on a cancer cell isEphA2.

Eph receptor tyrosine kinases (Ephs) belong to a large group of receptortyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosineresidues. Ephs and their membrane bound ephrin ligands (ephrins) controlcell positioning and tissue organization (Poliakov et al. (2004) DevCell 7, 465-80). Functional and biochemical Eph responses occur athigher ligand oligomerization states (Stein et al. (1998) Genes Dev 12,667-678).

Among other patterning functions, various Ephs and ephrins have beenshown to play a role in vascular development. Knockout of EphB4 andephrin-B2 results in a lack of the ability to remodel capillary bedsinto blood vessels (Poliakov et al., supra) and embryonic lethality.Persistent expression of some Eph receptors and ephrins has also beenobserved in newly-formed, adult micro-vessels (Brantley-Sieders et al.(2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

The de-regulated re-emergence of some ephrins and their receptors inadults also has been observed to contribute to tumor invasion,metastasis and neo-angiogenesis (Nakamoto et al. (2002) Microsc Res Tech59, 58-67; Brantley-Sieders et al., supra). Furthermore, some Eph familymembers have been found to be over-expressed on tumor cells from avariety of human tumors (Brantley-Sieders et al., supra); Marme (2002)Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humansis encoded by the EPHA2 gene.

EphA2 is upregulated in multiple cancers in man, often correlating withdisease progression, metastasis and poor prognosis e.g.: breast(Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010)Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6,e24426), lung (Brannan et al (2009) Cancer Prey Res (Phila) 2,1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al(2013) J Thorac Oncol. 8, 301-308), gastric (Nakamura et al (2005)Cancer Sci. 96, 42-47; Yuan et al (2009) Dig Dis Sci 54, 2410-2417),pancreatic (Mudali et al (2006) Clin Exp Metastasis 23, 357-365),prostate (Walker-Daniels et al (1999) Prostate 41, 275-280), liver (Yanget al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al(2005) Mol Cancer Res. 3, 541-551; Li et al (2010) Tumour Biol. 31,477-488).

The full role of EphA2 in cancer progression is still not definedalthough there is evidence for interaction at numerous stages of cancerprogression including tumour cell growth, survival, invasion andangiogenesis. Downregulation of EphA2 expression suppresses tumourcancer cell propagation (Binda et al (2012) Cancer Cell 22, 765-780),whilst EphA2 blockade inhibits VEGF induced cell migration (Hess et al(2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng etal (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40)and metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19,1884-1886).

An antibody drug conjugate to EphA2 has been shown to significantlydiminish tumour growth in rat and mouse xenograft models (Jackson et al(2008) Cancer Research 68, 9367-9374) and a similar approach has beentried in man although treatment had to be discontinued for treatmentrelated adverse events (Annunziata et al (2013) Invest New drugs 31,77-84).

In one embodiment, the first peptide ligand comprises an EphA2 bindingbicyclic peptide ligand.

Suitable examples of EphA2 binding bicyclic peptide ligands aredisclosed in WO 2019/122860, WO 2019/122861 and WO 2019/122863, thepeptides of which are incorporated herein by reference.

In one embodiment, the EphA2 binding bicyclic peptide is selected fromany of the peptides of SEQ ID NOS: 10 to 51 described herein.

In an alternative embodiment, the component present on a cancer cell isPD-L1.

Programmed cell death 1 ligand 1 (PD-L1) is a 290 amino acid type Itransmembrane protein encoded by the CD274 gene on mouse chromosome 19and human chromosome 9. PD-L1 expression is involved in evasion ofimmune responses involved in chronic infection, e.g., chronic viralinfection (including, for example, HIV, HBV, HCV and HTLV, amongothers), chronic bacterial infection (including, for example,Helicobacter pylori, among others), and chronic parasitic infection(including, for example, Schistosoma mansoni). PD-L1 expression has beendetected in a number of tissues and cell types including T-cells,B-cells, macrophages, dendritic cells, and nonhaematopoietic cellsincluding endothelial cells, hepatocytes, muscle cells, and placenta.

PD-L1 expression is also involved in suppression of anti-tumour immuneactivity. Tumours express antigens that can be recognised by hostT-cells, but immunologic clearance of tumours is rare. Part of thisfailure is due to immune suppression by the tumour microenvironment.PD-L1 expression on many tumours is a component of this suppressivemilieu and acts in concert with other immunosuppressive signals. PD-L1expression has been shown in situ on a wide variety of solid tumoursincluding breast, lung, colon, ovarian, melanoma, bladder, liver,salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck(Brown J A et al. 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat.Med. 8:793-800; Hamanishi J, et al. 2007 Proc. Natl. Acad. Sci. USA104:3360-65; Strome S E et al. 2003 Cancer Res. 63:6501-5; Inman B A etal. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res.10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother.56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101: 17174-79; Wu C et al. 2006Acta Histochem. 108:19-24). In addition, the expression of the receptorfor PD-L1, Programmed cell death protein 1 (also known as PD-1 andCD279) is upregulated on tumour infiltrating lymphocytes, and this alsocontributes to tumour immunosuppression (Blank C et al. 2003 Immunol.171:4574-81). Most importantly, studies relating PD-L1 expression ontumours to disease outcome show that PD-L1 expression stronglycorrelates with unfavourable prognosis in kidney, ovarian, bladder,breast, gastric, and pancreatic cancer (Hamanishi J et al. 2007 Proc.Natl. Acad. Sci. USA 104:3360-65; Inman B A et al. 2007 Cancer109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100;Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi Tet al. 2007 Clin. Cancer Res. 13:2151-57; Thompson R H et al. 2004 Proc.Natl. Acad. Sci. USA 101:17174-79; Wu C et al. 2006 Acta Histochem.108:19-24). In addition, these studies suggest that higher levels ofPD-L1 expression on tumours may facilitate advancement of tumour stageand invasion into deeper tissue structures.

The PD-1 pathway can also play a role in haematologic malignancies.PD-L1 is expressed on multiple myeloma cells but not on normal plasmacells (Liu J et al. 2007 Blood 110:296-304). PD-L1 is expressed on someprimary T-cell lymphomas, particularly anaplastic large cell T lymphomas(Brown J A et al, 2003 Immunol. 170:1257-66). PD-1 is highly expressedon the T-cells of angioimmunoblastic lymphomas, and PD-L1 is expressedon the associated follicular dendritic cell network (Dorfman D M et al.2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominantHodgkin lymphoma, the T-cells associated with lymphocytic or histiocytic(L&H) cells express PD-1. Microarray analysis using a readout of genesinduced by PD-1 ligation suggests that tumour-associated T-cells areresponding to PD-1 signals in situ in Hodgkin lymphoma (Chemnitz J M etal. 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T-cellsin HTLV-1-mediated adult T-cell leukaemia and lymphoma (Shimauchi T etal. 2007 Int. J. Cancer 121: 2585-90). These tumour cells arehyporesponsive to TCR signals.

Studies in animal models demonstrate that PD-L1 on tumours inhibitsT-cell activation and lysis of tumour cells and in some cases leads toincreased tumour-specific T-cell death (Dong H et al. 2002 Nat. Med.8:793-800; Hirano F et al. 2005 Cancer Res. 65:1089-96).Tumour-associated APCs can also utilise the PD-1:PD-L1 pathway tocontrol antitumour T-cell responses. PD-L1 expression on a population oftumour-associated myeloid DCs is upregulated by tumour environmentalfactors (Curiel T J et al. 2003 Nat. Med. 9:562-67). Plasmacytoiddendritic cells (DCs) in the tumour-draining lymph node of B16 melanomaexpress IDO, which strongly activates the suppressive activity ofregulatory T-cells. The suppressive activity of IDO-treated regulatoryT-cells required cell contact with IDO-expressing DCs (Sharma M D et al.2007 Clin. Invest. 117:2570-82).

In one embodiment, the first peptide ligand comprises a PD-L1 bindingbicyclic peptide ligand.

Suitable examples of PD-L1 binding bicyclic peptide ligands aredisclosed in GB Patent Application Nos. 1905631.6 and 1904622.6, thepeptides of which are incorporated herein by reference.

In one embodiment, the PD-L1 binding bicyclic peptide is selected fromany of the peptides of SEQ ID NOS: 1 to 9 described herein.

In an alternative embodiment, the component present on a cancer cell isprostate-specific membrane antigen (PSMA).

Prostate-specific membrane antigen (PSMA) (also known as Glutamatecarboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I(NAALADase I) and NAAG peptidase) is an enzyme that in humans is encodedby the FOLH1 (folate hydrolase 1) gene. Human GCPII contains 750 aminoacids and weighs approximately 84 kDa.

Human PSMA is highly expressed in the prostate, roughly a hundred timesgreater than in most other tissues. In some prostate cancers, PSMA isthe second-most upregulated gene product, with an 8- to 12-fold increaseover levels in noncancerous prostate cells. Because of this highexpression, PSMA is being developed as potential biomarker for therapyand imaging of some cancers. In human prostate cancer, the higherexpressing tumors are associated with quicker time to progression and agreater percentage of patients suffering relapse.

In one embodiment, the first peptide ligand comprises a PSMA bindingbicyclic peptide ligand.

Suitable examples of PSMA binding bicyclic peptide ligands are disclosedin GB Patent Application Nos 1820325.7 and 1912723.2 and PCT PatentApplication No. PCT/EP2019/066273, the peptides of which areincorporated herein by reference.

Second Peptide Ligands

References herein to the term “immune cell” includes any cell within theimmune system. Suitable examples include white blood cells, such aslymphocytes (e.g. T lymphocytes or T cells, B cells or natural killercells). In one embodiment, the T cell is CD8 or CD4. In a furtherembodiment, the T cell is CD8. Other examples of immune cells includedendritic cells, follicular dendritic cells and granulocytes.

In one embodiment, the component present on an immune cell is CD137.

CD137 is a member of the tumour necrosis factor (TNF) receptor family.Its alternative names are tumour necrosis factor receptor superfamilymember 9 (TNFRSF9), 4-IBB and induced by lymphocyte activation (ILA).CD137 can be expressed by activated T cells, but to a larger extent onCD8+ than on CD4+ T cells. In addition, CD137 expression is found ondendritic cells, follicular dendritic cells, natural killer cells,granulocytes and cells of blood vessel walls at sites of inflammation.One characterized activity of CD137 is its costimulatory activity foractivated T cells. Crosslinking of CD137 enhances T cell proliferation,IL-2 secretion, survival and cytolytic activity. Further, it can enhanceimmune activity to eliminate tumours in mice.

CD137 is a T-cell costimulatory receptor induced on TCR activation (Namet al., Curr. Cancer Drug Targets, 5:357-363 (2005); Waits et al., Annu.Rev, Immunol., 23:23-68 (2005)). In addition to its expression onactivated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+regulatory T cells, natural killer (NK) and NK-T cells, monocytes,neutrophils, and dendritic cells. Its natural ligand, CD137L, has beendescribed on antigen-presenting cells including B cells,monocyte/macrophages, and dendritic cells (Watts et al. Annu. Rev.Immunol, 23:23-68 (2005)). On interaction with its ligand, CD137 leadsto increased TCR-induced T-cell proliferation, cytokine production,functional maturation, and prolonged CD8+ T-cell survival (Nam et al,Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et d-I., Annu. Rev.Immunol, 23:23-68 (2005)).

Signalling through CD137 by either CD137L or agonistic monoclonalantibodies (mAbs) against CD137 leads to increased TCR-induced T cellproliferation, cytokine production and functional maturation, andprolonged CD8+ T cell survival. These effects result from: (1) theactivation of the NF-κB, c-Jun NH2-terminal kinase/stress-activatedprotein kinase (JNK/SAPK), and p38 mitogen-activated protein kinase(MAPK) signalling pathways, and (2) the control of anti-apoptotic andcell cycle-related gene expression.

Experiments performed in both CD137 and CD137L-deficient mice haveadditionally demonstrated the importance of CD137 costimulation in thegeneration of a fully competent T cell response.

IL-2 and IL-15 activated NK cells express CD137, and ligation of CD137by agonistic mAbs stimulates NK cell proliferation and IFN-γ secretion,but not their cytolytic activity.

Furthermore, CD137-stimulated NK cells promote the expansion ofactivated T cells in vitro.

In accordance with their costimulatory function, agonist mAbs againstCD137 have been shown to promote rejection of cardiac and skinallografts, eradicate established tumours, broaden primary antiviralCD8+ T cell responses, and increase T cell cytolytic potential. Thesestudies support the view that CD137 signalling promotes T cell functionwhich may enhance immunity against tumours and infection.

In one embodiment, the second peptide ligand comprises a CD137 bindingbicyclic peptide ligand.

Suitable examples of CD137 binding bicyclic peptide ligands aredisclosed in WO 2019/025811, the peptides of which are incorporatedherein by reference.

In one embodiment, the CD137 binding bicyclic peptide is selected fromany of the peptides of SEQ ID NOS: 67 to 84 described herein.

Linkers It will be appreciated that the first peptide ligand may beconjugated to the second peptide ligand via any suitable linker.Typically the design of said linker will be such that the two Bicyclicpeptides are presented in such a manner that they can bind unencumberedto their respective targets either alone or while simultaneously bindingto both target receptors. Additionally, the linker should permit bindingto both targets simultaneously while maintaining an appropriate distancebetween the target cells that would lead to the desired functionaloutcome. The properties of the linker may be modulated to increaselength, rigidity or solubility to optimise the desired functionaloutcome. The linker may also be designed to permit the attachment ofmore than one Bicycle to the same target. Increasing the valency ofeither binding peptide may serve to increase the affinity of theheterotandem for the target cells or may help to induce oligomerisationof one or both of the target receptors.

In one embodiment, the linker is selected from the following sequences:-PEG5- and TCA-[PEG₁₀]₃.

Structural representations of these linkers are detailed below:

Heterotandem Complexes

In one specific embodiment, the first peptide ligand comprises a PD-L1binding bicyclic peptide ligand attached to a TATA scaffold, the secondpeptide ligand comprises a CD137 binding bicyclic peptide ligandattached to a TATA scaffold and said heterotandem complex is selectedfrom the complexes listed in Table A:

TABLE A (PD-L1:CD137; 1:1) Complex PD-L1 BCY Attachment CD137 BCYAttachment No. No. Point Linker No. Point BCY12229 BCY11865 Lys9 Peg5BCY8928 dLys(PYA)4 BCY12230 BCY11866 Lys2 Peg5 BCY8928 dLys(PYA)4BCY12231 BCY11867 Lys7 Peg5 BCY8928 dLys(PYA)4 BCY12232 BCY11868 Lys8Peg5 BCY8928 dLys(PYA)4 BCY12242 BCY11869 Lys11 Peg5 BCY8928 dLys(PYA)4BCY12375 BCY10861 Lys(PYA)9 Peg5 BCY12023 dLys4 BCY12663 BCY12479 C-termLys Peg5 BCY8928 dLys(PYA)4 BCY12796 BCY12477 C-term Lys Peg5 BCY8928dLys(PYA)4 BCY12021 BCY10861 Lys(PYA)9 Peg5 BCY11144 dLys4

In one embodiment, the heterotandem bicyclic peptide complex is selectedfrom: BCY12375 and BCY12021.

In one specific embodiment, the first peptide ligand comprises an EphA2binding bicyclic peptide ligand attached to a TATA scaffold, the secondpeptide ligand comprises a CD137 binding bicyclic peptide ligandattached to a TATA scaffold and said heterotandem complex is selectedfrom the complexes listed in Table B:

TABLE B (EphA2:CD137; 1:1) Complex EphA2 BCY Attachment CD137 BCYAttachment No. No. Point Linker No. Point BCY12233 BCY11813 N-term PYAPeg5 BCY8920 dLys4 BCY12234 BCY11814 C-term Lys(PYA) Peg5 BCY8920 dLys4BCY12235 BCY11815 Lys(PYA) 8 Peg5 BCY8920 dLys4 BCY12236 BCY11816Lys(PYA)2 Peg5 BCY8920 dLys4 BCY12237 BCY11817 Lys(PYA)7 Peg5 BCY8920dLys4 BCY12711 BCY9594 N-terminus Peg5 BCY12143 dLys (PYA)4 BCY12712BCY9594 N-terminus Peg5 BCY12149 dLys (PYA)4 BCY12713 BCY9594 N-terminusPeg5 BCY12147 dLys (PYA)4 BCY12714 BCY9594 N-terminus Peg5 BCY12145 dLys(PYA)4 BCY12715 BCY9594 N-terminus Peg5 BCY12146 dLys (PYA)4 BCY12717BCY9594 N-terminus Peg5 BCY12352 dLys (PYA)4 BCY12718 BCY9594 N-terminusPeg5 BCY12353 dLys (PYA)4 BCY12719 BCY9594 N-terminus Peg5 BCY12354 dLys(PYA)4 BCY12720 BCY9594 N-terminus Peg5 BCY12360 dLys (PYA)4 BCY12961BCY12734 C-term Lys Peg5 BCY8928 dLys (PYA)4 BCY12962 BCY12735 Lys8 Peg5BCY8928 dLys (PYA)4 BCY12963 BCY12736 Lys2 Peg5 BCY8928 dLys (PYA)4BCY12964 BCY12737 Lys7 Peg5 BCY8928 dLys (PYA)4 BCY12965 BCY12738N-terminus Peg5 BCY8928 dLys (PYA)4 BCY12966 BCY12739 N-terminus Peg5BCY8928 dLys (PYA)4 BCY13029 BCY12854 N-terminus Peg5 BCY8928 dLys(PYA)4 BCY13030 BCY12855 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13031BCY12856 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13032 BCY12857N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13033 BCY12858 N-terminus Peg5BCY8928 dLys (PYA)4 BCY13034 BCY12859 N-terminus Peg5 BCY8928 dLys(PYA)4 BCY13035 BCY12860 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13036BCY12861 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13037 BCY12862N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13038 BCY12863 N-terminus Peg5BCY8928 dLys (PYA)4 BCY13039 BCY12864 N-terminus Peg5 BCY8928 dLys(PYA)4 BCY13040 BCY12865 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13041BCY12866 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13141 BCY12856N-terminus Peg5 BCY12353 dLys (PYA)4 BCY13142 BCY9594 N-terminus Peg5BCY13137 dLys (PYA)4 BCY13143 BCY12856 N-terminus Peg5 BCY13137 dLys(PYA)4 BCY13250 BCY13116 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13251BCY13117 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13252 BCY13118N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13253 BCY13119 N-terminus Peg5BCY8928 dLys (PYA)4 BCY13254 BCY13120 C-term dLys Peg5 BCY8928 dLys(PYA)4 BCY13255 BCY13121 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13256BCY13122 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13257 BCY13123N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13258 BCY13124 N-terminus Peg5BCY8928 dLys (PYA)4 BCY13260 BCY13126 N-terminus Peg5 BCY8928 dLys(PYA)4 BCY13261 BCY13127 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13262BCY13128 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13264 BCY13130N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13265 BCY13131 N-terminus Peg5BCY8928 dLys (PYA)4 BCY13266 BCY13132 N-terminus Peg5 BCY8928 dLys(PYA)4 BCY13268 BCY13134 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13269BCY13135 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13340 BCY12865N-terminus Peg5 BCY12353 dLys (PYA)4 BCY13342 BCY12860 N-terminus Peg5BCY12353 dLys (PYA)4

In one embodiment, the heterotandem bicyclic peptide complex is selectedfrom: BCY13035, BCY13040, BCY13253, BCY13254, BCY13340 and BCY13342.

In one specific embodiment, the first peptide ligand comprises aNectin-4 binding bicyclic peptide ligand attached to a TATA scaffold,the second peptide ligand comprises a CD137 binding bicyclic peptideligand attached to a TATA scaffold and said heterotandem complex isselected from the complexes listed in Table C:

TABLE C (Nectin-4:CD137; 1:1) Complex Nectin-4 BCY Attachment CD137 BCYAttachment No. No. Point Linker No. Point BCY11616 BCY8116 N-terminusPeg5 BCY7744 dLys(PYA)4 BCY12238 BCY12024 dLys3 Peg5 BCY8928 dLys(PYA)4BCY12377 BCY8116 N-terminus Peg5 BCY12143 dLys(PYA)4 BCY12379 BCY8116N-terminus Peg5 BCY12149 dLys(PYA)4 BCY12572 BCY8116 N-terminus Peg5BCY12352 dLys(PYA)4 BCY12573 BCY8116 N-terminus Peg5 BCY12353 dLys(PYA)4BCY12574 BCY8116 N-terminus Peg5 BCY12354 dLys(PYA)4 BCY12575 BCY8116N-terminus Peg5 BCY12360 dLys(PYA)4 BCY12576 BCY12363 dLys3 Peg5 BCY8928dLys(PYA)4 BCY12577 BCY12364 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12578BCY12365 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12579 BCY12366 dLys3 Peg5BCY8928 dLys(PYA)4 BCY12580 BCY12367 dLys3 Peg5 BCY8928 dLys(PYA)4BCY12581 BCY12368 N-terminus Peg5 BCY8928 dLys(PYA)4 BCY12582 BCY12369N-terminus Peg5 BCY8928 dLys(PYA)4 BCY12583 BCY12370 N-terminus Peg5BCY8928 dLys(PYA)4 BCY12584 BCY12371 dLys3 Peg5 BCY8928 dLys(PYA)4BCY12585 BCY12384 N-terminus Peg5 BCY8928 dLys(PYA)4 BCY12709 BCY8116N-terminus Peg5 BCY12381 dLys(PYA)4 BCY12710 BCY8116 N-terminus Peg5BCY12382 dLys(PYA)4 BCY11468 BCY11016 N-term PYA TCA-[Peg10]3 BCY8928dLys(PYA)4 BCY11618 BCY11143 N-term PYA Peg5 BCY8920 dLys4 BCY11776BCY8116 N-terminus Peg5 BCY11144 C-term Dap(PYA) BCY11860 BCY11143N-term PYA Peg5 BCY8920 dLys4 BCY12020 BCY11016 N-term PYA Peg5 BCY11144C-term Dap(PYA) BCY12661 BCY11015 N-term PYA Peg5 BCY12023 dLys4BCY12969 BCY8116 N-terminus Peg5 BCY12358 dLys(PYA)4

In one embodiment, the heterotandem bicyclic peptide complex is selectedfrom: BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661 andBCY12969.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

Nomenclature

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added tothe left or right side of the sequence, separated by a hyphen. Forexample, an N-terminal βAla-Sar10-Ala tail would be denoted as:

βAla-Sar10-A-(SEQ ID NO:X).

Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3),1362-1373, it is envisaged that the peptide sequences disclosed hereinwould also find utility in their retro-inverso form. For example, thesequence is reversed (i.e. N-terminus becomes C-terminus and vice versa)and their stereochemistry is likewise also reversed (i.e. D-amino acidsbecome L-amino acids and vice versa). For the avoidance of doubt,references to amino acids either as their full name or as their aminoacid single or three letter codes are intended to be represented hereinas L-amino acids unless otherwise stated. If such an amino acid isintended to be represented as a D-amino acid then the amino acid will beprefaced with a lower case d within square parentheses, for example[dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalentlybound to a molecular scaffold. Typically, such peptides comprise two ormore reactive groups (i.e. cysteine residues) which are capable offorming covalent bonds to the scaffold, and a sequence subtended betweensaid reactive groups which is referred to as the loop sequence, since itforms a loop when the peptide is bound to the scaffold. In the presentcase, the peptides comprise at least three reactive groups selected fromcysteine, 3-mercaptopropionic acid and/or cysteamine and form at leasttwo loops on the scaffold.

Reactive Groups

The molecular scaffold of the invention may be bonded to the polypeptidevia functional or reactive groups on the polypeptide. These aretypically formed from the side chains of particular amino acids found inthe polypeptide polymer. Such reactive groups may be a cysteine sidechain, a lysine side chain, or an N-terminal amine group or any othersuitable reactive group, such as penicillamine. Details of suitablereactive groups may be found in WO 2009/098450.

Examples of reactive groups of natural amino acids are the thiol groupof cysteine, the amino group of lysine, the carboxyl group of aspartateor glutamate, the guanidinium group of arginine, the phenolic group oftyrosine or the hydroxyl group of serine. Non-natural amino acids canprovide a wide range of reactive groups including an azide, aketo-carbonyl, an alkyne, a vinyl, or an aryl halide group. The aminoand carboxyl group of the termini of the polypeptide can also serve asreactive groups to form covalent bonds to a molecular scaffold/molecularcore.

The polypeptides of the invention contain at least three reactivegroups. Said polypeptides can also contain four or more reactive groups.The more reactive groups are used, the more loops can be formed in themolecular scaffold.

In a preferred embodiment, polypeptides with three reactive groups aregenerated. Reaction of said polypeptides with a molecularscaffold/molecular core having a three-fold rotational symmetrygenerates a single product isomer. The generation of a single productisomer is favourable for several reasons. The nucleic acids of thecompound libraries encode only the primary sequences of the polypeptidebut not the isomeric state of the molecules that are formed uponreaction of the polypeptide with the molecular core. If only one productisomer can be formed, the assignment of the nucleic acid to the productisomer is clearly defined. If multiple product isomers are formed, thenucleic acid cannot give information about the nature of the productisomer that was isolated in a screening or selection process. Theformation of a single product isomer is also advantageous if a specificmember of a library of the invention is synthesized. In this case, thechemical reaction of the polypeptide with the molecular scaffold yieldsa single product isomer rather than a mixture of isomers.

In another embodiment, polypeptides with four reactive groups aregenerated. Reaction of said polypeptides with a molecularscaffold/molecular core having a tetrahedral symmetry generates twoproduct isomers. Even though the two different product isomers areencoded by one and the same nucleic acid, the isomeric nature of theisolated isomer can be determined by chemically synthesizing bothisomers, separating the two isomers and testing both isomers for bindingto a target ligand.

In one embodiment of the invention, at least one of the reactive groupsof the polypeptides is orthogonal to the remaining reactive groups. Theuse of orthogonal reactive groups allows the directing of saidorthogonal reactive groups to specific sites of the molecular core.Linking strategies involving orthogonal reactive groups may be used tolimit the number of product isomers formed. In other words, by choosingdistinct or different reactive groups for one or more of the at leastthree bonds to those chosen for the remainder of the at least threebonds, a particular order of bonding or directing of specific reactivegroups of the polypeptide to specific positions on the molecularscaffold may be usefully achieved.

In another embodiment, the reactive groups of the polypeptide of theinvention are reacted with molecular linkers wherein said linkers arecapable to react with a molecular scaffold so that the linker willintervene between the molecular scaffold and the polypeptide in thefinal bonded state.

In some embodiments, amino acids of the members of the libraries or setsof polypeptides can be replaced by any natural or non-natural aminoacid. Excluded from these exchangeable amino acids are the onesharbouring functional groups for cross-linking the polypeptides to amolecular core, such that the loop sequences alone are exchangeable. Theexchangeable polypeptide sequences have either random sequences,constant sequences or sequences with random and constant amino acids.The amino acids with reactive groups are either located in definedpositions within the polypeptide, since the position of these aminoacids determines loop size.

In one embodiment, a polypeptide with three reactive groups has thesequence (X)_(l)Y(X)_(m)Y(X)_(n)Y(X)_(o), wherein Y represents an aminoacid with a reactive group, X represents a random amino acid, m and nare numbers between 3 and 6 defining the length of interveningpolypeptide segments, which may be the same or different, and l and oare numbers between 0 and 20 defining the length of flanking polypeptidesegments.

Alternatives to thiol-mediated conjugations can be used to attach themolecular scaffold to the peptide via covalent interactions.Alternatively these techniques may be used in modification or attachmentof further moieties (such as small molecules of interest which aredistinct from the molecular scaffold) to the polypeptide after they havebeen selected or isolated according to the present invention—in thisembodiment then clearly the attachment need not be covalent and mayembrace non-covalent attachment. These methods may be used instead of(or in combination with) the thiol mediated methods by producing phagethat display proteins and peptides bearing unnatural amino acids withthe requisite chemical reactive groups, in combination small moleculesthat bear the complementary reactive group, or by incorporating theunnatural amino acids into a chemically or recombinantly synthesisedpolypeptide when the molecule is being made after theselection/isolation phase. Further details can be found in WO2009/098450 or Heinis et al., Nat Chem Biol 2009, 5 (7), 502-7.

In one embodiment, the reactive groups are selected from cysteine,3-mercaptopropionic acid and/or cysteamine residues.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of thisinvention, and references to peptide ligands include the salt forms ofsaid ligands.

The salts of the present invention can be synthesized from the parentcompound that contains a basic or acidic moiety by conventional chemicalmethods such as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. One particular salt is the hydrochloride salt.Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO), then a salt may be formed with anorganic or inorganic base, generating a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).Examples of some suitable substituted ammonium ions are those derivedfrom: methylamine, ethylamine, diethylamine, propylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the compounds of the invention contain an amine function, thesemay form quaternary ammonium salts, for example by reaction with analkylating agent according to methods well known to the skilled person.Such quaternary ammonium compounds are within the scope of theinvention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligandsas defined herein are within the scope of the present invention.Examples of such suitable modified derivatives include one or moremodifications selected from: N-terminal and/or C-terminal modifications;replacement of one or more amino acid residues with one or morenon-natural amino acid residues (such as replacement of one or morepolar amino acid residues with one or more isosteric or isoelectronicamino acids; replacement of one or more non-polar amino acid residueswith other non-natural isosteric or isoelectronic amino acids); additionof a spacer group; replacement of one or more oxidation sensitive aminoacid residues with one or more oxidation resistant amino acid residues;replacement of one or more amino acid residues with an alanine,replacement of one or more L-amino acid residues with one or moreD-amino acid residues; N-alkylation of one or more amide bonds withinthe bicyclic peptide ligand; replacement of one or more peptide bondswith a surrogate bond; peptide backbone length modification;substitution of the hydrogen on the alpha-carbon of one or more aminoacid residues with another chemical group, modification of amino acidssuch as cysteine, lysine, glutamate/aspartate and tyrosine with suitableamine, thiol, carboxylic acid and phenol-reactive reagents so as tofunctionalise said amino acids, and introduction or replacement of aminoacids that introduce orthogonal reactivities that are suitable forfunctionalisation, for example azide or alkyne-group bearing amino acidsthat allow functionalisation with alkyne or azide-bearing moieties,respectively.

In one embodiment, the modified derivative comprises an N-terminaland/or C-terminal modification. In a further embodiment, wherein themodified derivative comprises an N-terminal modification using suitableamino-reactive chemistry, and/or C-terminal modification using suitablecarboxy-reactive chemistry. In a further embodiment, said N-terminal orC-terminal modification comprises addition of an effector group,including but not limited to a cytotoxic agent, a radiochelator or achromophore.

In a further embodiment, the modified derivative comprises an N-terminalmodification. In a further embodiment, the N-terminal modificationcomprises an N-terminal acetyl group. In this embodiment, the N-terminalcysteine group (the group referred to herein as C_(i)) is capped withacetic anhydride or other appropriate reagents during peptide synthesisleading to a molecule which is N-terminally acetylated. This embodimentprovides the advantage of removing a potential recognition point foraminopeptidases and avoids the potential for degradation of the bicyclicpeptide.

In an alternative embodiment, the N-terminal modification comprises theaddition of a molecular spacer group which facilitates the conjugationof effector groups and retention of potency of the bicyclic peptide toits target.

In a further embodiment, the modified derivative comprises a C-terminalmodification. In a further embodiment, the C-terminal modificationcomprises an amide group. In this embodiment, the C-terminal cysteinegroup (the group referred to herein as C_(iii)) is synthesized as anamide during peptide synthesis leading to a molecule which isC-terminally amidated. This embodiment provides the advantage ofremoving a potential recognition point for carboxypeptidase and reducesthe potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of oneor more amino acid residues with one or more non-natural amino acidresidues. In this embodiment, non-natural amino acids may be selectedhaving isosteric/isoelectronic side chains which are neither recognisedby degradative proteases nor have any adverse effect upon targetpotency.

Alternatively, non-natural amino acids may be used having constrainedamino acid side chains, such that proteolytic hydrolysis of the nearbypeptide bond is conformationally and sterically impeded. In particular,these concern proline analogues, bulky sidechains, Cα-disubstitutedderivatives (for example, aminoisobutyric acid, Aib), and cyclo aminoacids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of aspacer group. In a further embodiment, the modified derivative comprisesthe addition of a spacer group to the N-terminal cysteine (C_(i)) and/orthe C-terminal cysteine (C_(iii)).

In one embodiment, the modified derivative comprises replacement of oneor more oxidation sensitive amino acid residues with one or moreoxidation resistant amino acid residues. In a further embodiment, themodified derivative comprises replacement of a tryptophan residue with anaphthylalanine or alanine residue. This embodiment provides theadvantage of improving the pharmaceutical stability profile of theresultant bicyclic peptide ligand.

In one embodiment, the modified derivative comprises replacement of oneor more charged amino acid residues with one or more hydrophobic aminoacid residues. In an alternative embodiment, the modified derivativecomprises replacement of one or more hydrophobic amino acid residueswith one or more charged amino acid residues. The correct balance ofcharged versus hydrophobic amino acid residues is an importantcharacteristic of the bicyclic peptide ligands. For example, hydrophobicamino acid residues influence the degree of plasma protein binding andthus the concentration of the free available fraction in plasma, whilecharged amino acid residues (in particular arginine) may influence theinteraction of the peptide with the phospholipid membranes on cellsurfaces. The two in combination may influence half-life, volume ofdistribution and exposure of the peptide drug, and can be tailoredaccording to the clinical endpoint. In addition, the correct combinationand number of charged versus hydrophobic amino acid residues may reduceirritation at the injection site (if the peptide drug has beenadministered subcutaneously).

In one embodiment, the modified derivative comprises replacement of oneor more L-amino acid residues with one or more D-amino acid residues.This embodiment is believed to increase proteolytic stability by sterichindrance and by a propensity of D-amino acids to stabilise β-turnconformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of anyamino acid residues and substitution with alanines. This embodimentprovides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serveto deliberately improve the potency or stability of the peptide. Furtherpotency improvements based on modifications may be achieved through thefollowing mechanisms:

-   -   Incorporating hydrophobic moieties that exploit the hydrophobic        effect and lead to lower off rates, such that higher affinities        are achieved;    -   Incorporating charged groups that exploit long-range ionic        interactions, leading to faster on rates and to higher        affinities (see for example Schreiber et al, Rapid,        electrostatically assisted association of proteins (1996),        Nature Struct. Biol. 3, 427-31); and    -   Incorporating additional constraint into the peptide, by for        example constraining side chains of amino acids correctly such        that loss in entropy is minimal upon target binding,        constraining the torsional angles of the backbone such that loss        in entropy is minimal upon target binding and introducing        additional cyclisations in the molecule for identical reasons.        (for reviews see Gentilucci et al, Curr. Pharmaceutical Design,        (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem        (2009), 16, 4399-418).

Isotopic Variations

The present invention includes all pharmaceutically acceptable(radio)isotope-labeled peptide ligands of the invention, wherein one ormore atoms are replaced by atoms having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberusually found in nature, and peptide ligands of the invention, whereinmetal chelating groups are attached (termed “effector”) that are capableof holding relevant (radio)isotopes, and peptide ligands of theinvention, wherein certain functional groups are covalently replacedwith relevant (radio)isotopes or isotopically labelled functionalgroups.

Examples of isotopes suitable for inclusion in the peptide ligands ofthe invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T),carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, suchas ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³N and¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur,such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga,yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies, and to clinically assessthe presence and/or absence of the Nectin-4 target on diseased tissues.The peptide ligands of the invention can further have valuablediagnostic properties in that they can be used for detecting oridentifying the formation of a complex between a labelled compound andother molecules, peptides, proteins, enzymes or receptors. The detectingor identifying methods can use compounds that are labelled withlabelling agents such as radioisotopes, enzymes, fluorescent substances,luminous substances (for example, luminol, luminol derivatives,luciferin, aequorin and luciferase), etc. The radioactive isotopestritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly usefulfor this purpose in view of their ease of incorporation and ready meansof detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention cangenerally be prepared by conventional techniques known to those skilledin the art or by processes analogous to those described in theaccompanying Examples using an appropriate isotopically-labeled reagentin place of the non-labeled reagent previously employed.

Molecular Scaffold

Molecular scaffolds are described in, for example, WO 2009/098450 andreferences cited therein, particularly WO 2004/077062 and WO2006/078161.

As noted in the foregoing documents, the molecular scaffold may be asmall molecule, such as a small organic molecule.

In one embodiment, the molecular scaffold may be a macromolecule. In oneembodiment, the molecular scaffold is a macromolecule composed of aminoacids, nucleotides or carbohydrates.

In one embodiment, the molecular scaffold comprises reactive groups thatare capable of reacting with functional group(s) of the polypeptide toform covalent bonds.

The molecular scaffold may comprise chemical groups which form thelinkage with a peptide, such as amines, thiols, alcohols, ketones,aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides,anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

In one embodiment, the molecular scaffold may comprise or may consist ofhexahydro-1,3,5-triazine, especially1,3,5-Triacryloylhexahydro-1,3,5-triazine (TATA), or a derivativethereof.

The molecular scaffold of the invention contains chemical groups thatallow functional groups of the polypeptide of the encoded library of theinvention to form covalent links with the molecular scaffold. Saidchemical groups are selected from a wide range of functionalitiesincluding amines, thiols, alcohols, ketones, aldehydes, nitriles,carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides,maleimides, azides, alkyl halides and acyl halides.

Scaffold reactive groups that could be used on the molecular scaffold toreact with thiol groups of cysteines are alkyl halides (or also namedhalogenoalkanes or haloalkanes).

Examples include bromomethylbenzene or iodoacetamide. Other scaffoldreactive groups that are used to selectively couple compounds tocysteines in proteins are maleimides, αβ unsaturated carbonyl containingcompounds and α-halomethylcarbonyl containing compounds. Examples ofmaleimides which may be used as molecular scaffolds in the inventioninclude: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene,tris-(maleimido)benzene. An example of an αβ unsaturated carbonylcontaining compound is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)(Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). Anexample of an α-halomethylcarbonyl containing compound isN,N′,N″-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine isalso a natural amino acid which has a similar reactivity to cysteine andcan be used for the same reactions. Thus, wherever cysteine ismentioned, it is typically acceptable to substitute selenocysteineunless the context suggests otherwise.

Synthesis

The peptides of the present invention may be manufactured syntheticallyby standard techniques followed by reaction with a molecular scaffold invitro. When this is performed, standard chemistry may be used. Thisenables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. Such methods could beaccomplished using conventional chemistry such as that disclosed inTimmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. In one embodiment, thesesteps are carried out on the end product polypeptide/conjugate made bychemical synthesis.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus or within the loops using orthogonallyprotected lysines (and analogues) using standard solid phase or solutionphase chemistry. Standard (bio)conjugation techniques may be used tointroduce an activated or activatable N- or C-terminus. Alternativelyadditions may be made by fragment condensation or native chemicalligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteinsby Native Chemical Ligation. Science 266:776-779), or by enzymes, forexample using subtiligase as described in (Chang et al. Proc Natl AcadSci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic &Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages6000-6003).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptides to dissociate from each otheronce within the reducing environment of the cell. In this case, themolecular scaffold (e.g. TATA) could be added during the chemicalsynthesis of the first peptide so as to react with the three cysteinegroups; a further cysteine or thiol could then be appended to the N orC-terminus of the first peptide, so that this cysteine or thiol onlyreacted with a free cysteine or thiol of the second peptides, forming adisulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of twobicyclic and bispecific macrocycles, potentially creating atetraspecific molecule.

Furthermore, addition of other functional groups or effector groups maybe accomplished in the same manner, using appropriate chemistry,coupling at the N- or C-termini or via side chains. In one embodiment,the coupling is conducted in such a manner that it does not block theactivity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand as defined hereinin combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand/or buffered media. Parenteral vehicles include sodium chloridesolution, Ringers dextrose, dextrose and sodium chloride and lactatedRingers. Suitable physiologically-acceptable adjuvants, if necessary tokeep a polypeptide complex in suspension, may be chosen from thickenerssuch as carboxymethylcellulose, polyvinylpyrrolidone, gelatin andalginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringers dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum andimmunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the proteinligands of the present invention, or even combinations of selectedpolypeptides according to the present invention having differentspecificities, such as polypeptides selected using different targetligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, the peptide ligands of the invention canbe administered to any patient in accordance with standard techniques.The administration can be by any appropriate mode, includingparenterally, intravenously, intramuscularly, intraperitoneally,transdermally, via the pulmonary route, or also, appropriately, bydirect infusion with a catheter. Preferably, the pharmaceuticalcompositions according to the invention will be administered byinhalation. The dosage and frequency of administration will depend onthe age, sex and condition of the patient, concurrent administration ofother drugs, counterindications and other parameters to be taken intoaccount by the clinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the peptide ligandsdescribed herein may be used extracorporeally or in vitro selectively tokill, deplete or otherwise effectively remove a target cell populationfrom a heterogeneous collection of cells. Blood from a mammal may becombined extracorporeally with the selected peptide ligands whereby theundesired cells are killed or otherwise removed from the blood forreturn to the mammal in accordance with standard techniques.

Therapeutic Uses

According to a further aspect of the invention, there is provided aheterotandem bicyclic peptide complex as defined herein for use inpreventing, suppressing or treating cancer.

Examples of cancers (and their benign counterparts) which may be treated(or inhibited) include, but are not limited to tumours of epithelialorigin (adenomas and carcinomas of various types includingadenocarcinomas, squamous carcinomas, transitional cell carcinomas andother carcinomas) such as carcinomas of the bladder and urinary tract,breast, gastrointestinal tract (including the esophagus, stomach(gastric), small intestine, colon, rectum and anus), liver(hepatocellular carcinoma), gall bladder and biliary system, exocrinepancreas, kidney, lung (for example adenocarcinomas, small cell lungcarcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomasand mesotheliomas), head and neck (for example cancers of the tongue,buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands,nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum,vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (forexample thyroid follicular carcinoma), adrenal, prostate, skin andadnexae (for example melanoma, basal cell carcinoma, squamous cellcarcinoma, keratoacanthoma, dysplastic naevus); haematologicalmalignancies (i.e. leukemias, lymphomas) and premalignant haematologicaldisorders and disorders of borderline malignancy includinghaematological malignancies and related conditions of lymphoid lineage(for example acute lymphocytic leukemia [ALL], chronic lymphocyticleukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma[DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma,T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas,Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy ofuncertain significance, plasmacytoma, multiple myeloma, andpost-transplant lymphoproliferative disorders), and haematologicalmalignancies and related conditions of myeloid lineage (for exampleacute myelogenousleukemia [AML], chronic myelogenousleukemia [CML],chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome,myeloproliferative disorders such as polycythaemia vera, essentialthrombocythaemia and primary myelofibrosis, myeloproliferative syndrome,myelodysplastic syndrome, and promyelocyticleukemia); tumours ofmesenchymal origin, for example sarcomas of soft tissue, bone orcartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas,rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas,Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioidsarcomas, gastrointestinal stromal tumours, benign and malignanthistiocytomas, and dermatofibrosarcomaprotuberans; tumours of thecentral or peripheral nervous system (for example astrocytomas, gliomasand glioblastomas, meningiomas, ependymomas, pineal tumours andschwannomas); endocrine tumours (for example pituitary tumours, adrenaltumours, islet cell tumours, parathyroid tumours, carcinoid tumours andmedullary carcinoma of the thyroid); ocular and adnexal tumours (forexample retinoblastoma); germ cell and trophoblastic tumours (forexample teratomas, seminomas, dysgerminomas, hydatidiform moles andchoriocarcinomas); and paediatric and embryonal tumours (for examplemedulloblastoma, neuroblastoma, Wilms tumour, and primitiveneuroectodermal tumours); or syndromes, congenital or otherwise, whichleave the patient susceptible to malignancy (for example XerodermaPigmentosum).

In a further embodiment, the cancer is selected from a hematopoieticmalignancy such as selected from: non-Hodgkin's lymphoma (NHL),Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocyticleukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T celllymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL),Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).

References herein to the term “prevention” involves administration ofthe protective composition prior to the induction of the disease.“Suppression” refers to administration of the composition after aninductive event, but prior to the clinical appearance of the disease.“Treatment” involves administration of the protective composition afterdisease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels. The invention is further described below with reference to thefollowing examples.

EXAMPLES

In general, the heterotandem bicyclic peptide complexes of the inventionmay be prepared in accordance with the following general method:

A mixture of Bicycle 1 (1.0 eq.) and NHS-PEG5-N3 (1.6 eq.) is dissolvedin MeCN/H₂O (1:1), and the pH of the solution adjusted to 8 by dropwiseaddition of NaHCO₃ (0.1 M). The reaction mixture is stirred at 30° C.for 2 hr then concentrated under reduced pressure to remove solvent. Theresidue is then purified by prep-HPLC to give intermediate 2.

A mixture of intermediate 2 (1.0 eq) and Bicycle2 (1.0 eq) are dissolvedin t-BuOH/H₂O (1:1), and then CuSO₄ (1.0 eq), VcNa (2.3 eq), and THPTA(1.0 eq) are added. Finally, 0.2 M NH₄HCO₃ is added to adjust pH to 8.The reaction mixture is stirred at 40° C. for 16 hr under N₂ atmosphere.The reaction mixture was directly purified by prep-HPLC.

More detailed experimental for selected heterotandem bicyclic peptidecomplexes of the invention are provided herein below:

Example 1: Synthesis of BCY12375

Procedure for Preparation of Palmitic Acid—PEG10-N₃

A mixture of Palmitic acid (100.0 mg, 282.89 μmol, 1.0 eq.), compound 2(150.0 mg, 284.84 μmol, 1.0 eq.), and DIEA (74.5 mg, 574.11 μmol, 100.0μL, 2.0 eq.) was dissolved in DMF (2 mL). The reaction mixture wasstirred at 30° C. for 2 hr. LC-MS showed compound 1 was consumedcompletely and one main peak with desired m/z (MW: 765.03, observed m/z:765.22) was detected. The reaction mixture was concentrated underreduced pressure to remove solvent and produced a residue. The residuewas then purified by prep-HPLC (neutral condition). Palmiticacid—PEG10-N₃ (79.0 mg, 99.41 μmol, 35.14% yield, 96.27% purity) wasobtained as a white solid.

Procedure for Preparation of Palmitic Acid-PEG10-BCY12023

A mixture of compound 3 (50.0 mg, 22.07 μmol, 1.0 eq.), compound 2 (17.0mg, 22.22 μmol, 1.0 eq.), and THPTA (10.0 mg, 23.02 μmol, 1.0 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ for3 times), and then CuSO₄ (0.4 M, 56.0 μL, 1.0 eq.) and VcNa (10.0 mg,50.48 μmol, 2.3 eq.) were added under N₂. The pH of this solution wasadjusted to 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O),and the solution turned to light yellow. The reaction mixture wasstirred at 40° C. for 2 hr under N₂ atmosphere. LC-MS showed Palmiticacid—PEG10-N₃ was remand and one main peak with desired m/z (calculatedMW: 3030.60, observed m/z: 1010.35 ([M/3+H]+)) was detected. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by prep-HPLC (TFAcondition), and Palmitic acid—PEG10-BCY12023 (43.0 mg, 13.97 μmol,63.30% yield, 98.46% purity) was obtained as a white solid.

Procedure for Preparation of Palmitic Acid—PEG10-BCY12023-PEG5-N₃

A mixture of compound 5 (43.0 mg, 14.19 μmol, 1.0 eq.), compound 6 (10.0mg, 23.13 μmol, 1.6 eq.) was dissolved in MeCN/H₂O (1:1, 1 mL), and thenthe pH of this solution was adjusted to 8 by dropwise addition of NaHCO₃(0.1 M). The reaction mixture was stirred at 30° C. for 2 hr. LC-MSshowed compound 5 was consumed completely and one main peak with desiredm/z (MW: 3347.94, observed m/z: 1673.7 ([(M/2+H⁺]), 1115.9 ([(M/3+H⁺]))was detected. The reaction mixture was concentrated under reducedpressure to remove solvent and produced a residue. The residue was thenpurified by prep-HPLC (neutral condition). Palmiticacid—PEG10-BCY12023-PEG5-N₃ (16.0 mg, 4.43 μmol, 31.25% yield, 92.78%purity) was obtained as a white solid.

Procedure for Preparation of BCY12375

A mixture of compound 7 (8.0 mg, 2.39 μmol, 1.0 eq.), compound 8 (6.5mg, 2.39 μmol, 1.0 eq.), and THPTA (1.1 mg, 2.53 μmol, 1.0 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ for3 times), and then CuSO₄ (0.4 M, 6.0 μL, 1.0 eq.) and VcNa (1.0 mg, 5.05μmol, 2.1 eq.) were added under N₂. The pH of this solution was adjustedto 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 40°C. for 16 hr under N₂ atmosphere. LC-MS showed compound 7 was remand andone main peak with desired m/z (calculated MW: 6064.08, observed m/z:1516.4 ([M/4+H]⁺), 1212.8 ([M/5+H]⁺)) was detected. The reaction mixturewas filtered and concentrated under reduced pressure to give a residue.The crude product was purified by prep-HPLC (TFA condition), andBCY12375 (6.2 mg, 0.99 μmol, 41.62% yield, 97.27% purity) was obtainedas a white solid.

Example 2: Synthesis of BCY12021

Procedure for Preparation of Palmitic Acid—PEG10-BCY11144

A mixture of compound 3 (160.0 mg, 69.45 μmol, 1.0 eq.), compound 4(56.0 mg, 72.20 μmol, 1.0 eq.), and THPTA (35.0 mg, 80.55 μmol, 1.1 eq.)was dissolved in t-BuOH/H₂O (1:1, 2 mL, pre-degassed and purged with N₂for 3 times), and then CuSO₄ (0.4 M, 56.0 μL, 1.0 eq.) and VcNa (30.0mg, 151.43 μmol, 2.2 eq.) were added under N₂. The pH of this solutionwas adjusted to 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1t-BuOH/H₂O), and the solution turned to light yellow. The reactionmixture was stirred at 40° C. for 16 hr under N₂ atmosphere. LC-MSshowed one main peak with desired m/z (calculated MW: 3068.70, observedm/z: 1533.81 ([M/2+H]⁺), 1023.43 ([M/3+H]⁺)) was detected. The reactionmixture was filtered and concentrated under reduced pressure to give aresidue. The crude product was purified by prep-HPLC (TFA condition),and Palmitic acid—PEG10-BCY11144 (150.0 mg, 46.83 μmol, 67.42% yield,95.80% purity) was obtained as a white solid.

Procedure for Preparation of Palmitic Acid—PEG10-BCY11144-PEG5-N₃

A mixture of compound 5 (47.0 mg, 15.32 μmol, 1.0 eq.), compound 6 (7.0mg, 16.19 μmol, 1.0 eq.), and DIEA (3.0 mg, 22.97 μmol, 4.0 μL, 1.5 eq.)was dissolved in DMF (1 mL). The reaction mixture was stirred at 30° C.for 2 hr. LC-MS showed compound 5 was consumed completely and one mainpeak with desired m/z (MW: 3386.03, observed m/z: 1693.21 ([M/2+H]⁺),1129.13 ([M/3+H]⁺)) was detected. The reaction mixture was concentratedunder reduced pressure to remove solvent and produced a residue. Theresidue was then purified by prep-HPLC (neutral condition). Palmiticacid—PEG10-BCY11144-PEG5-N₃ (20.0 mg, 5.72 μmol, 37.33% yield, 96.79%purity) was obtained as a white solid.

Procedure for Preparation of BCY12021

A mixture of compound 7 (10.0 mg, 2.95 μmol, 1.0 eq.), compound 8 (8.2mg, 3.02 μmol, 1.0 eq.), and THPTA (1.5 mg, 3.45 μmol, 1.1 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ 3times), and then CuSO₄ (0.4 M, 8.0 μL, 1.0 eq.) and VcNa (1.5 mg, 7.57μmol, 2.5 eq.) were added under N₂. The pH of this solution was adjustedto 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 40°C. for 16 hr under N₂ atmosphere. LC-MS showed one main peak withdesired m/z (calculated MW: 6102.17, observed m/z: 1525.17 ([M/4+H]⁺),1221.3 ([M/5+H]⁺)). The reaction mixture was filtered and concentratedunder reduced pressure to give a residue. The crude product was purifiedby prep-HPLC (TFA condition), and BCY12021 (6.6 mg, 1.02 μmol, 34.62%yield, 94.54% purity) was obtained as a white solid.

Example 3: Synthesis of BCY11468

Procedure for Preparation of COM113

A mixture of compound 1 (50.0 mg, 124.4 μmol, 1.0 eq), EDCl (95.4 mg,497.7 μmol, 4.0 eq), HOBt (55.5 mg, 410.6 μmol, 3.3 eq), and DMAP (15.2mg, 124.4 μmol, 1.0 eq) was dissolved in 2 mL DMF, and then DIEA (134.9mg, 1.04 mmol, 181.8 μL, 8.4 eq) was added to generate a homogenoussolution. Next, compound 2 (200.0 mg, 379.8 μmol, 3.05 eq) dissolved inDMF (2 mL) was added to this solution dropwise. The reaction mixture wasstirred at 30° C. for 16 hr. LC-MS showed compound 1 was consumedcompletely and one main peak with desired m/z (MW: 1891.19, observedm/z: 945.8600 ([M/2+H⁺]) and 612.4400 ([(M-3H₂O)/3+H⁺])) was detected.The reaction mixture was directly purified by prep-HPLC (TFA condition),resulting in COM113 (161 mg, 85.67 μmol, 68% yield) as a yellow oilafter lyophilization.

Procedure for Preparation of COM113-BCY8928

COM113 (50.0 mg, 26.44 μmol, 1.0 eq) and BCY8928 (53.0 mg, 23.9 μmol,0.9 eq) were first dissolved in 2 mL of t-BuOH/H₂O (1:1), and then CuSO₄(0.4 M, 66.1 μL, 1.0 eq), VcNa (10.5 mg, 53.0 μmol, 2.0 eq) and THPTA(23.0 mg, 52.93 μmol, 2.0 eq) was added. Finally, 1 M NH₄HCO₃ was addedto adjust pH to 8. All solvents were degassed and purged with N₂ for 3times. The reaction mixture was stirred at 30° C. for 16 hr under N₂atmosphere. LC-MS showed one main peak with desired m/z (calculated MW:4108.77 observed m/z: 1369.97 ([M/3+H]⁺)). The reaction mixture waspurified by prep-HPLC (TFA condition) and Compound 2 (14.0 mg, 3.21μmol, 12.14% yield, 94.16% purity) was obtained as a white solid.

Procedure for Preparation of Palmitic Acid NHS Ester

To a solution of palmitic acid (500 mg, 1.95 mmol, 586.85 μL, 1.0 eq),1-hydroxypyrrolidine-2,5-dione (250 mg, 2.17 mmol, 1.11 eq) in DCM (5mL) was added with EDCl (747.60 mg, 3.90 mmol, 2.0 eq). The mixture wasstirred at 30° C. for 16 hr. TLC indicated Reactant 1 was consumedcompletely and one new spot formed. The reaction was clean according toTLC. The reaction mixture was filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO2, DCM: MeOH=0 to 100:1). The desired product wasdried to obtain Palmitic acid NHS ester (0.68 g, 1.92 mmol, 98.65%yield) as a white solid.

Procedure for Preparation of Palmitic Acid-Propargylalanine

To a solution of compound 3 (120 mg, 339.47 μmol, 1.0 eq) and compound 4(57.60 mg, 509.20 μmol, 1.5 eq) in DMF (6 mL) was added DIEA (131.62 mg,1.02 mmol, 177.39 μL, 3.0 eq) and DMAP (41.47 mg, 339.47 μmol, 1.0 eq).The mixture was stirred at 40° C. for 16 hr. LC-MS showed Reactant 3 wasconsumed completely and one main peak with desired m/z or desired masswas detected. The reaction mixture was filtered and concentrated underreduced pressure to give a residue. The residue was purified byprep-HPLC (TFA condition). Palmitic acid-Propargylalanine (90 mg, 256.03μmol, 75.42% yield) was obtained as a white solid.

Procedure for Preparation of COM113-BCY8928-Palmitic Acid

Compound 2 (14.0 mg, 3.41 μmol, 1.0 eq) and Compound 3 (1.1 mg, 3.13μmol, 0.9 eq) were first dissolved in 2 mL of t-BuOH/H₂O (1:1), and thenCuSO₄ (0.4 M, 10.0 μL, 1.1 eq), VcNa (2.0 mg, 10.1 μmol, 2.9 eq) andTHPTA (2.0 mg, 4.6 μmol, 1.3 eq) was added. Finally 0.2 M NH₄HCO₃ wasadded to adjust pH to 8. All solvents here were degassed and purged withN₂ for 3 times. The reaction mixture was stirred at 35° C. for 16 hrunder N₂ atmosphere. LC-MS showed one main peak with desired m/z(calculated MW: 4460.29, observed m/z: 1486.92 ([M/3+H]⁺), 1115.58([M/4+H]⁺), 895.83 ([M/5+H]⁺)). The reaction mixture was purified byprep-HPLC (TFA condition) and Compound 4 (5.9 mg, 1.28 μmol, 37.66%yield, 97.0% purity) was obtained as a white solid.

Procedure for Preparation of BCY11468

Compound 4 (5.9 mg, 1.32 μmol, 1.0 eq) and BCY11016 (3.0 mg, 1.29 μmol,1 eq) were first dissolved in 2 mL of t-BuOH/H₂O (1:1), and then CuSO₄(0.4 M, 8.0 μL, 2.4 eq), VcNa 2.0 mg, 7.6 eq) and THPTA (2.0 mg, 3.5 eq)was added. Finally, 1 M NH₄HCO₃ was added to adjust pH to 8. Allsolvents were degassed and purged with N₂ 3 times. The reaction mixturewas stirred at 30° C. for 16 hr under N₂ atmosphere. LC-MS showed onemain peak with desired m/z (calculated MW: 6783.93, observed m/z: 1131.7([M/6+H]⁺)). The reaction mixture was purified by prep-HPLC (TFAcondition) and BCY11468 (2.2 mg, 0.312 μmol, 23.57% yield, 96.16%purity) was obtained as a white solid.

Example 4: Synthesis of BCY11618

Procedure for Preparation of BCY8920-PEG5-N₃

A mixture of BCY8920 (50.0 mg, 23.39 μmol, 1.0 eq.), compound 2 (10.2mg, 23.51 μmol, 1.01 eq.) and NaHCO₃ (2.0 mg, 24.8 μmol, 1.0 eq.) wasdissolved in MeCN/H₂O (1:1, 2 mL). The reaction mixture was stirred at40° C. for 2 hr, until LC-MS showed BCY8920 was consumed completely andone main peak with desired m/z (calculated MW: 2454.83, observed m/z:1227.67 ([M/2+1-1]⁺) and 818.74 ([M/3+1-1]⁺)) was detected. The reactionmixture was then concentrated under reduced pressure to remove solventand produced a residue, following by purification by prep-HPLC (TFAcondition). BCY8920-PEG5-N₃ (25 mg, 9.70 μmol, 41.47% yield, 95.26%purity) was obtained as a white solid.

Procedure for Preparation of BCY11143-dK(Palmitic Acid)

A mixture of BCY11143 (30.0 mg, 12.84 μmol, 1.0 eq.), compound 5 (5.0mg, 14.12 μmol, 1.1 eq.), DIEA (1.7 mg, 12.84 μmol, 2.2 μL, 1.0 eq.) andDMAP (1.6 mg, 12.84 μmol, 1.0 eq.) was dissolved in DMF. The reactionmixture was stirred at 40° C. for 2 hr under N₂ atmosphere. LC-MS showedone main peak with desired m/z (calculated MW: 2575.14, observed m/z:1287.68 ([M/2+H⁺])) was detected. The reaction mixture was filtered andconcentrated under reduced pressure to give a residue, which was thenpurified by prep-HPLC (TFA condition). BCY11143-dK(palmitic acid) (18.3mg, 6.95 μmol, 54.17% yield, 97.86% purity) was obtained as a whitesolid.

Procedure for Preparation of BCY11618

A mixture of compound 3 (5 mg, 2.04 μmol, 1.0 eq.), compound 6 (5.8 mg,2.3 μmol, 1.1 eq.), and THPTA (0.9 mg, 2.07 μmol, 1.0 eq.) was dissolvedin t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ for 3 times),and then CuSO₄ (0.4 M, 5.1 μL, 1.0 eq.) and VcNa (0.4 M, 5.1 μL, 1.0eq.) were added under N₂. The pH of this solution was adjusted to 8 bydropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and the solutionturned to light yellow. The reaction mixture was stirred at 40° C. for 6hr under N₂ atmosphere. LC-MS showed compound 3 was consumed completelyand one main peak with desired m/z (calculated MW: 5029.97, observedm/z: 1257.8 ([M/4+H]⁺) and 1006.6 ([M/5+H]⁺)) was detected. The reactionmixture was filtered and concentrated under reduced pressure to give aresidue. The crude product was purified by prep-HPLC (TFA condition),and BCY11618 (5.3 mg, 1.0 μmol, 49.15% yield, 95% purity) was obtainedas a white solid.

Example 5: Synthesis of BCY11776

Procedure for Preparation of BCY8116-Peg5-N₃

A mixture of BCY8116 (50.0 mg, 23.39 μmol, 1.0 eq.), compound 2 (10.2mg, 23.51 μmol, 1.01 eq.) and NaHCO₃ (2.0 mg, 24.8 μmol, 1.0 eq.) wasdissolved in MeCN/H₂O (1:1, 2 mL). The reaction mixture was stirred at25° C. for 1 hr until LC-MS showed BCY8116 was consumed completely andone main peak with desired m/z (calculated MW: 2454.83, observed m/z:1227.67 ([M/2+H⁺]), 818.74 ([M/3+H⁺])) was detected. The reactionmixture was then concentrated under reduced pressure to remove solventand produced a residue, following by purification by prep-HPLC (TFAcondition). Compound 3 (25.0 mg, 9.70 μmol, 41.47% yield, 95.26% purity)was obtained as a white solid.

Procedure for Preparation of Compound BCY11144-dK(Palmitic Acid)

A mixture of BCY11144 (50.0 mg, 21.7 μmol, 1.0 eq.), compound 5 (8.5 mg,23.87 μmol, 1.1 eq.), DIEA (2.81 mg, 21.7 μmol, 4.0 μL, 1.0 eq.) andDMAP (2.7 mg, 21.7 μmol, 1.0 eq.) was dissolved in DMF. The reactionmixture was stirred at 25° C. for 2 hr under N₂ atmosphere. LC-MS showedcompound 3 was consumed completely and one main peak with desired m/z(calculated MW: 2542.08, observed m/z: 1271.7 ([M/2+H⁺])) was detected.The reaction mixture was filtered and concentrated under reducedpressure to give a residue, which was then purified by prep-HPLC (TFAcondition). Compound 6 (18.3 mg, 6.95 μmol, 54.17% yield, 96.68% purity)was obtained as a white solid.

Procedure for Preparation of BCY11776

A mixture of compound 3 (10 mg, 4.0 μmol, 1.0 eq.), compound 6 (11.2 mg,4.4 μmol, 1.1 eq.), and THPTA (1.8 mg, 1.0 eq.) was dissolved int-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ for 3 times), andthen CuSO₄ (0.4 M, 5.1 μL, 1 eq.) and VcNa (0.4 M, 5.1 μL, 1 eq.) wereadded under N₂. The pH of this solution was adjusted to 8 by dropwiseaddition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and the solution turnedto light yellow. The reaction mixture was stirred at 40° C. for 6 hrunder N₂ atmosphere. LC-MS showed compound 3 was consumed completely andone main peak with desired m/z (calculated MW: 5031.9, observed m/z:1258.52 ([M/4+H⁺]), 1006.7 ([M/5+H⁺])) was detected. The reactionmixture was filtered and concentrated under reduced pressure to give aresidue. The crude product was purified by prep-HPLC (TFA condition),and BCY11776 (12.5 mg, 2.4 μmol, 60.11% yield, 96.6% purity) wasobtained as a white solid.

Example 6: Synthesis of BCY11860

Procedure for Preparation of BCY8920-Peg5-BCY11143

A mixture of BCY8920-PEG5-N₃ (20.0 mg, 8.15 μmol, 1.0 eq.), compound 2(21.0 mg, 8.96 μmol, 1.1 eq.), and THPTA (0.4 M, 21.0 μL, 1.0 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ for3 times), and then CuSO₄ (0.4 M, 21.0 μL, 1.0 eq.) and VcNa (0.4 M, 21.0μL, 1.0 eq.) were added under N₂. The pH of this solution was adjustedto 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 40°C. for 4 hr under N₂ atmosphere. LC-MS showed compound 1 was consumedcompletely and one main peak with desired m/z (calculated MW: 4791.56,observed m/z: 1597.28 ([M/3+H]⁺), 1198.18 ([M/4+H]⁺)) was detected. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by prep-HPLC (TFAcondition), and BCY8920-Peg5-BCY11143 (22.5 mg, 4.25 μmol, 52.13% yield,90.44% purity) was obtained as a white solid.

Procedure for Preparation of BCY11860

A mixture of compound 3 (5.0 mg, 1.04 μmol, 1.0 eq.), compound 4 (1.08mg, 1.15 μmol, 1.1 eq.), and DIEA (0.4 M, 1.04 μmol, 3.0 μL, 1.0 eq.)and DMAP (0.2 mg, 1.04 μmol, 1.0 eq.) was dissolved in DMF (1.0 mL). Thereaction mixture was stirred at 30° C. for 2 hr. LC-MS showed compound 3was consumed completely and one main peak with desired m/z (MW: 5617.56,observed m/z: 1404.56 ([(M/4+H⁺])) was detected. The reaction mixturewas concentrated under reduced pressure to remove solvent and produced aresidue. The residue was then purified by prep-HPLC (neutral condition).BCY11860 (2.9 mg, 0.48 μmol, 45.86% yield, 92.70% purity) was obtainedas a white solid.

Example 7: Synthesis of BCY12020

Procedure for Preparation of Palmitic Acid-PEG10-N₃

A mixture of Palmitic acid-NHS (100.0 mg, 282.89 μmol, 1.0 eq.),compound 2 (150.0 mg, 284.84 μmol, 1.0 eq.), and DIEA (74.5 mg, 574.11μmol, 100.0 μL, 2.0 eq.) was dissolved in DMF (2 mL). The reactionmixture was stirred at 30° C. for 2 hr. LC-MS showed compound 1 wasconsumed completely and one main peak with desired m/z (MW: 765.03,observed m/z: 765.22) was detected. The reaction mixture wasconcentrated under reduced pressure to remove solvent and produced aresidue. The residue was then purified by prep-HPLC (neutral condition).Palmitic acid-PEG10-N₃ (79.0 mg, 99.41 μmol, 35.14% yield, 96.27%purity) was obtained as a white solid.

Procedure for Preparation of Palmitic Acid-PEG10-BCY11144

A mixture of compound 3 (160.0 mg, 69.45 μmol, 1.0 eq.), compound 2(56.0 mg, 72.20 μmol, 1.0 eq.), and THPTA (35.0 mg, 80.55 μmol, 1.1 eq.)was dissolved in t-BuOH/H₂O (1:1, 2 mL, pre-degassed and purged with N₂3 times), and then CuSO₄ (0.4 M, 56.0 μL, 1.0 eq.) and VcNa (30.0 mg,151.43 μmol, 2.2 eq.) were added under N₂. The pH of this solution wasadjusted to 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O),and the solution turned to light yellow. The reaction mixture wasstirred at 40° C. for 16 hr under N₂ atmosphere. LC-MS showed one mainpeak with desired m/z (calculated MW: 3068.70, observed m/z: 1533.81([M/2+H]⁺), 1023.43 ([M/3+H]⁺)). The reaction mixture was filtered andconcentrated under reduced pressure to give a residue. The crude productwas purified by prep-HPLC (TFA condition), and Palmiticacid-PEG10-BCY11144 (150.0 mg, 46.83 μmol, 67.42% yield, 95.80% purity)was obtained as a white solid.

Procedure for Preparation of Palmitic Acid-PEG10-BCY11144-PEG5-N₃

A mixture of compound 5 (47.0 mg, 15.32 μmol, 1.0 eq.), compound 6 (7.0mg, 16.19 μmol, 1.1 eq.), and DIEA (3.0 mg, 22.97 μmol, 4.0 μL, 1.5 eq.)was dissolved in DMF (1 mL). The reaction mixture was stirred at 30° C.for 2 hr. LC-MS showed compound 5 was consumed completely and one mainpeak with desired m/z (MW: 3386.03, observed m/z: 1693.21 ([M/2+H]⁺),1129.13 ([M/3+H]⁺)) was detected. The reaction mixture was concentratedunder reduced pressure to remove solvent and produced a residue. Theresidue was then purified by prep-HPLC (neutral condition). Palmiticacid-PEG10-BCY11144-PEG5-N₃ (20.0 mg, 5.72 μmol, 37.33% yield, 96.79%purity) was obtained as a white solid.

Procedure for Preparation of BCY12020

A mixture of compound 7 (50.0 mg, 14.77 μmol, 1.0 eq.), compound 8 (35.0mg, 15.06 μmol, 1.0 eq.), and THPTA (10.0 mg, 23.02 μmol, 1.5 eq.) wasdissolved in t-BuOH/H₂O (1:1, 2 mL, pre-degassed and purged with N₂ for3 times), and then CuSO₄ (0.4 M, 38.0 μL, 1.0 eq.) and VcNa (6.5 mg,32.81 μmol, 2.2 eq.) were added under N₂. The pH of this solution wasadjusted to 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O),and the solution turned to light yellow. The reaction mixture wasstirred at 40° C. for 16 hr under N₂ atmosphere. LC-MS showed one mainpeak with desired m/z (calculated MW: 5709.68, observed m/z: 1902.80([M/3+H]⁺), 1427.56 ([M/4+H]⁺)). The reaction mixture was filtered andconcentrated under reduced pressure to give a residue. The crude productwas purified by prep-HPLC (TFA condition), and BCY12020 (54.8 mg, 9.49μmol, 64.24% yield, 98.83% purity) was obtained as a white solid.

Example 8: Synthesis of BCY12661

Procedure for Preparation of Compound 2

The peptide was synthesized using standard Fmoc chemistry. DCM was addedto a reaction vessel containing Chlorotrityl resin (1 mmol, 0.91 g, 1.10mmol/g) and Fmoc-Lys(N₃)—OH (1 eq, 395.4 mg, 1 mmol) with N₂ bubbling.DIEA (4.0 eq) was added dropwise and mixed for 2 hours. MeOH (2 mL) wasthen added and mixed for 30 min. The resin was drained and washed withDMF 5 times. Fmoc deprotection was performed by addition of 20%piperidine/DMF and mixing for 30 min. The resin was drained and washedwith DMF 5 times. For chain elongation, Fmoc-amino acid solution wasadded and mixed for 30 sec first, then add activation buffer (containingHBTU and DIEA in DMF) was added and stirred for 1 hr with continuous N₂bubbling. Deprotection and coupling was repeated until the peptide wascomplete.

# Materials Coupling reagents 1 Fmoc-Lys(N3) -OH (1 eq) DIEA(4.0 eq) 2Fmoc-γGlu(OtBu)-OH (3 eq) HBTU(2.85 eq) and DIEA(6.0 eq) 3 Palmitic acid(3 eq) HBTU(2.85 eq) and DIEA(6.0 eq)

After last amino acid coupling, the resin was washed with MeOH 3 times,and then dried under vacuum. 10 ml of cleavage cocktail (95% TFA/2.5%TIS/2.5% H₂O) was added to the flask containing the side-chain protectedpeptide at room temperature and this was stirred for 1 hour. The resinwas filtered and the filtrate concentrated to remove the solvent. Thecrude peptide was lyophilized to give the final product Compound 2(palmitic acid azide) (200 mg, 97.78% purity, 37.06% yield). CalculatedMW: 539.72, observed m/z: 540.4 ([M+H]⁺).

Procedure for Preparation of BCY12023-palmitic acid azide

A mixture of compound 1 (40.0 mg, 17.66 μmol, 1.0 eq.), compound 2 (9.5mg, 17.66 μmol, 1.0 eq.), and THPTA (8.0 mg, 17.66 μmol, 1.0 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ for3 times), and then CuSO₄ (0.4 M, 45.0 μL, 1.0 eq.) and VcNa (8.0 mg,35.33 μmol, 2.0 eq.) were added under N₂. The pH of this solution wasadjusted to 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O),and the solution turned to light yellow. The reaction mixture wasstirred at 40° C. for 4 hr under N₂ atmosphere. LC-MS showed compound 1was consumed completely and one main peak with desired m/z (calculatedMW: 2804.30, observed m/z: 1402.8 ([M/2+1-1]⁺), 935.9 ([M/3+H]⁺)) wasdetected. The reaction mixture was filtered and concentrated underreduced pressure to give a residue. The crude product was purified byprep-HPLC (TFA condition), and BCY12023-palmitic acid azide (35.0 mg,12.11 μmol, 68.54% yield, 97.00% purity) was obtained as a white solid.

Procedure for Preparation of BCY12023-Palmitic Acid-PEG5-N₃

A mixture of compound 3 (35.0 mg, 12.48 μmol, 1.0 eq.), compound 4 (5.4mg, 12.48 μmol, 1.0 eq.) was dissolved in MeCN/H₂O (1:1, 1 mL), and thenthe pH of this solution was adjusted to 8 by dropwise addition of NaHCO₃(0.1 M). The reaction mixture was stirred at 30° C. for 2 hr. LC-MSshowed compound 3 was consumed completely and one main peak with desiredm/z (MW: 3121.63, observed m/z: 1561.2 ([(M/2+H⁺])) was detected. Thereaction mixture was concentrated under reduced pressure to removesolvent and produced a residue. The residue was then purified byprep-HPLC (neutral condition). BCY12023-palmitic acid-PEG5-N₃ (11.4 mg,3.46 μmol, 27.71% yiled, 94.70% purity) was obtained as a white solid.

Procedure for Preparation of BCY12661

A mixture of compound 5 (11.4 mg, 3.65 μmol, 1.0 eq.), compound 6 (8.3mg, 3.65 μmol, 1.0 eq.), and THPTA (1.6 mg, 3.65 μmol, 1.0 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ 3times), and then CuSO₄ (0.4 M, 10.0 μL, 1.1 eq.) and VcNa (1.5 mg, 7.30μmol, 2.0 eq.) were added under N₂. The pH of this solution was adjustedto 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 40°C. for 4 hr under N₂ atmosphere. LC-MS showed compound 3 was consumedcompletely and one main peak with desired m/z (calculated MW: 5374.21,observed m/z: 1344.5 ([M/4+H]⁺)) was detected. The reaction mixture wasfiltered and concentrated under reduced pressure to give a residue. Thecrude product was purified by prep-HPLC (TFA condition), and BCY12661(9.8 mg, 19.63 μmol, 48.73% yield, 97.60% purity) was obtained as awhite solid.

Example 9: Synthesis of BCY12969

General Procedure for Preparation of Compound 1

The peptide was synthesized using standard Fmoc chemistry. DCM was addedto a reaction vessel containing chlorotrityl resin (1 mmol, 0.91 g, 1.1mmol/g) and Fmoc-γGlu(OtBu)-OH (0.425 mg, 1 mmol, 1 eq) and the mixturestirred with N₂ bubbling. DIEA (4.0 eq) was added dropwise and themixture was agitated for 2 hours. MeOH (4.6 mL) was then added and mixedfor 30 min. The resin was drained and washed with DMF 5 times. 20%piperidine/DMF was added to the resin and mixed for 30 minutes. Theresin was drained and washed with DMF 5 times. Fmoc-amino acid solutionwas added to the resin and mixed for 30 seconds, then activating agentand DIPEA was added and N₂ bubbled through the mixture for 1 hour.Deprotection and coupling steps were repeated with the followingreagents:

Note:

# Materials Coupling reagents 1 Fmoc-Glu-OtBu (1 eq) DIEA(4.0 eq) 2Fmoc-Glu-OtBu (3 eq) HBTU(2.85 eq) and DIEA(6.0 eq) 3 Palmitic acid (3eq) HBTU(2.85 eq) and DIEA(6.0 eq)

After coupling of Palmitic acid, the resin was washed 3 times with MeOHand then dried under vacuum. The peptide was cleaved from the resin byaddition of 20% HFIP/80% DCM at room temperature and the mixture stirredfor 1 hour. This procedure was repeated once more then the resin wasfiltered and the filtrate concentrated to remove the solvent. The crudepeptide was lyophilized to give the final product (280 mg, 84.80%purity, 44.67% yield). Calculated MW: 626.8, observed m/z: 627.4([M+H]⁺)

General Procedure for Preparation of Compound 3

To a solution of compound 2 (15.8 mg, 25.1 μmol, 1.1 eq) in DMF (0.5 mL)was added EDCl (4.4 mg, 22.8 μmol, 1.0 eq) and stirred for 10 min. ThenHOSu (2.9 mg, 25.1 μmol, 1.1 eq) and DIEA (8.8 mg, 68.5 μmol, 11.9 μL, 3eq) were added to the mixture. The mixture was stirred for 16 hr at 25°C. Then BCY12358 (50.0 mg, 22.8 μmol, 1.0 eq) in DMF (0.5 mL) was addedto the mixture and this was stirred at 25° C. for another 4 hr. LC-MSshowed BCY12358 was consumed completely and one main peak with desiredm/z (Calculated MW: 2798.42, observed m/z: 1399.6 [M/2+H]⁺) wasdetected. The reaction mixture was purified by prep-HPLC (A: 0.075% TFAin H₂O, B: ACN) to give compound 3 (21.9 mg, 7.83 μmol, 34.3% yield) asa white solid.

General Procedure for Preparation of Compound 5

A mixture of compound 4 (20.0 mg, 8.03 μmol, 1.0 eq), compound 3 (22.5mg, 8.03 μmol, 1.0 eq) and THPTA (4.0 mg, 9.21 μmol, 1.15 eq) in t-BuOH(0.5 mL) and H₂O (0.5 mL) was degassed and purged with N₂ 3 times, andthen CuSO₄ (0.4 M, 20.1 μL, 1.0 eq), VcNa (0.4 M, 40.2 μL, 2.0 eq) andNH₄HCO₃ (0.2 M, 80.4 μL, 2.0 eq) were added to the mixture. The mixturewas stirred at 30° C. for 2 hr under N₂ atmosphere. LC-MS showedcompound 4 was consumed completely and one main peak with desired m/z(Calculated MW: 5288.25, observed m/z: 1322.3 [M/4+H]⁺, 1763.8[M/3+1-1]⁺) was detected. EDTA (0.5 M, 20.0 μL) was added to thereaction mixture. The reaction mixture was concentrated under reducedpressure to give a crude product compound 5 (42.0 mg, crude) as graysolid and used into the next step without further purification.

General Procedure for Preparation of BCY12969

To a solution of compound 5 (42.0 mg, 8.22 μmol, 1.0 eq) in DCM (0.25mL) was added TFA (3.37 μmol, 0.25 mL, 458.6 eq) dropwise. The mixturewas stirred at 30° C. for 1 hr. LC-MS showed compound 5 was consumedcompletely and one main peak with desired m/z (Calculated MW: 5176.04,observed m/z: 1035.7 [M/5+H]⁺, 1294.9 [M/4+H]⁺, 1726.8 [M/3+1-1]⁺) wasdetected. The reaction mixture was concentrated under reduced pressureto give residue. The residue was purified by prep-HPLC (A: 0.075% TFA inH₂O, B: ACN) to give BCY12969 (2.6 mg, 0.48 μmol, 5.85% yield, 92.4%purity) as a white solid.

Example 10: Synthesis of BCY13035

Procedure for Preparation of BCY12860-PEG5-N₃

A mixture of BCY12860 (40.0 mg, 19.40 μmol, 1.0 eq.), compound 2 (10.0mg, 21.34 μmol, 1.1 eq.) was dissolved in MeCN/H₂O (1:1, 1 mL), and thenthe pH of this solution was adjusted to 8 by dropwise addition of NaHCO₃(0.1 M). The reaction mixture was stirred at 25° C. for 1 hr. LC-MSshowed a peak with desired m/z. The reaction mixture was concentratedunder reduced pressure to remove solvent and produced a residue. Theresidue was then purified by prep-HPLC (neutral condition).BCY12860-PEG5-N₃ (39.7 mg, 15.02 μmol, 77.41% yield, 90.0% purity) wasobtained as a white solid. MW: 2378.78, observed m/z: 1190.1([(M/2+H⁺]), 793.5 ([(M/3+H⁺]).

Procedure for Preparation of BCY13035

A mixture of compound 3 (39.7 mg, 16.69 μmol, 1.0 eq.), BCY8928 (41.0mg, 18.36 μmol, 1.1 eq.), and THPTA (0.4 M, 55 μL, 1.3 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ 3times), and then CuSO₄ (0.4 M, 55 μL, 1.3 eq.) and VcNa (0.4 M, 109 μL,2.6 eq.) were added under N₂. The pH of this solution was adjusted to 8by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 40°C. for 2 hr under N₂ atmosphere. LC-MS showed compound 3 was consumedcompletely and one main peak with desired m/z was detected. The reactionmixture was filtered and concentrated under reduced pressure to give aresidue. The crude product was purified by prep-HPLC (TFA condition),and BCY13035 (42.0 mg, 8.85 μmol, 53.04% yield, 96.41% purity) wasobtained as a white solid. Calculated MW: 4596.37, observed m/z: 1532.9([M/3+H]⁺), 1149.9 ([M/4+H]⁺).

Example 11: Synthesis of BCY13040

Procedure for Preparation of BCY12865-PEG5-N₃

BCY12865 (30.0 mg, 13.99 μmol, 1.0 eq) and compound 1 (6.1 mg, 14.11μmol, 1.01 eq), were dissolved in 1 mL of MeCN/H₂O (1:1), and then 1 MNaHCO₃ was added to adjust pH to 8. The mixture was stirred at 25° C.for 2 hr. LC-MS showed BCY12865 was consumed completely and one mainpeak with desired m/z was detected. The reaction mixture was purified byprep-HPLC (TFA condition) and compound 2 (15.6 mg, 6.32 μmol, 45.19%yield, 99.76% purity) was obtained as a white solid. Calculated MW:2461.87, observed m/z: 1231.5 ([M/2+H]⁺) and 821.3 ([M/3+H]⁺).

Procedure for Preparation of BCY13040

Compound 2 (15.6 mg, 6.34 μmol, 1.0 eq) and BCY8928 (14.5 mg, 6.54 μmol,1.03 eq) were first dissolved in 2 mL of t-BuOH/H₂O (1:1), and thenCuSO₄ (0.4 M, 16 μL, 1.01 eq), VcNa (3.0 mg, 15.14 μmol, 2.39 eq) andTHPTA (3 mg, 6.90 μmol, 1.09 eq) were added. Finally, 1 M NH₄HCO₃ wasadded to adjust the pH to 8. All solvents were degassed and purged withN₂ 3 times. The reaction mixture was stirred at 40° C. for 16 hr underN₂ atmosphere. LC-MS showed Compound 2 was consumed completely and onemain peak with desired m/z was detected. The reaction mixture waspurified by prep-HPLC (TFA condition) and BCY13040 (15.8 mg, 3.31 μmol,52.27% yield, 98.1% purity) was obtained as a white solid. CalculatedMW: 4679.45, observed m/z: 1560.8 ([M/3+H]⁺), 1170.9 ([M/4+H]⁺), 936.6([M/5+H]⁺).

Example 12: Synthesis of BCY13253

Procedure for Preparation of BCY13119-PEG5-N₃

A mixture of BCY13119 (35.0 mg, 17.20 μmol, 1.0 eq.), compound 2 (7.8mg, 18.06 μmol, 1.05 eq.) was dissolved in MeCN/H₂O (1:1, 1 mL), andthen the pH of this solution was adjusted to 8 by dropwise addition ofNaHCO₃ (0.1 M). The reaction mixture was stirred at 25° C. for 1 hr.LC-MS showed one main peak with desired m/z (MW: 2352.74, observed m/z:1177.4 ([(M/2+H⁺])) was detected. The reaction mixture was concentratedunder reduced pressure to remove solvent and produced a residue. Theresidue was then purified by prep-HPLC (neutral condition).BCY13119-PEG5-N₃ (25.7 mg, 9.97 μmol, 58.0% yield, 91.3% purity) wasobtained as a white solid.

Procedure for Preparation of BCY13253

A mixture of compound 3 (25.7 mg, 10.92 μmol, 1.0 eq.), compound 2 (26.6mg, 12.02 μmol, 1.1 eq.), and THPTA (5.7 mg, 13.11 μmol, 1.2 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ 3times), and then CuSO₄ (0.4 M, 33.0 μL, 1.2 eq.) and VcNa (5.2 mg, 26.21μmol, 2.4 eq.) were added under N₂. The pH of this solution was adjustedto 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 25°C. for 2 hr under N₂ atmosphere. LC-MS showed compound 3 was consumedcompletely and one main peak with desired m/z (calculated MW: 4570.32,observed m/z: 1143.4 ([M/4+1-1]⁺), 914.9 ([M/5+H]⁺)) was detected. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by prep-HPLC (TFAcondition), and BCY13253 (17.5 mg, 3.67 μmol, 33.58% yield, 95.8%purity) was obtained as a white solid.

Example 13: Synthesis of BCY13254

Procedure for Preparation of BCY13120-PEG5-N₃

A mixture of BCY13120 (40.0 mg, 17.92 μmol, 1.0 eq.), compound 2 (8.5mg, 19.72 μmol, 1.1 eq.) was dissolved in MeCN/H₂O (1:1, 1 mL), and thenthe pH of this solution was adjusted to 8 by dropwise addition of NaHCO₃(0.1 M). The reaction mixture was stirred at 25° C. for 1 hr. LC-MSshowed BCY13120 was consumed completely and one main peak with desiredm/z (MW: 2548.99, observed m/z: 1275.3 ([(M/2+H⁺])) was detected. Thereaction mixture was concentrated under reduced pressure to removesolvent and produced a residue. The residue was then purified byprep-HPLC (neutral condition). BCY13120-PEG5-N₃ (27.3 mg, 10.46 μmol,58.38% yield, 97.7% purity) was obtained as a white solid.

Procedure for Preparation of BCY13254

A mixture of compound 3 (27.3 mg, 10.71 μmol, 1.0 eq.), compound 2 (26.1mg, 11.78 μmol, 1.1 eq.), and THPTA (5.6 mg, 12.85 μmol, 1.2 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ 3times). CuSO₄ (0.4 M, 33.0 μL, 1.2 eq.) and VcNa (5.2 mg, 26.24 μmol,2.4 eq.) were added under N₂. The pH of this solution was adjusted to 8by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 25°C. for 2 hr under N₂ atmosphere. LC-MS showed compound 3 was consumedcompletely and one main peak with desired m/z (calculated MW: 4766.58,observed m/z: 1192.5 ([M/4+H]⁺), 954.1 ([M/5+H]⁺)) was detected. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by prep-HPLC (TFAcondition), and BCY13254 (36.5 mg, 7.49 μmol, 69.92% yield, 97.8%purity) was obtained as a white solid.

Example 14: Synthesis of BCY13340

Procedure for Preparation of BCY12865-PEG5-N₃

BCY12865 (50 mg, 23.32 μmol, 1.0 eq) and compound 1 (10.5 mg, 24.28μmol, 1.04 eq), were dissolved in 2 mL of MeCN/H₂O (1:1), 1 M NaHCO₃ wasadded to adjust pH to 8. And then the mixture was stirred at 25° C. for2 hr. LC-MS showed BCY12865 was consumed completely and one main peakwith desired m/z (calculated MW: 2461.87, observed m/z: 1231.6([M/2+H]⁺) and 821.4 ([M/3+H]⁺)) was detected. The reaction mixture waspurified by prep-HPLC (TFA condition) and compound 2 (31.5 mg, 12.62μmol, 54.14% yield, 98.66% purity) was obtained as a white solid.

Procedure for Preparation of BCY13340

A mixture of compound 2 (31.5 mg, 12.80 μmol, 1.0 eq.), BCY12353 (27 mg,12.92 μmol, 1.0 eq), and THPTA (5.7 mg, 13.12 μmol, 1.0 eq) wasdissolved in t-BuOH/H₂O (1:1, 2 mL, pre-degassed and purged with N₂ 3times), and then CuSO₄ (0.4 M, 32 μL, 1.0 eq) and VcNa (5.1 mg, 25.74μmol, 2.0 eq) were added under N₂. The pH of this solution was adjustedto 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned to light yellow. The reaction mixture was stirred at 40°C. for 1 hr under N₂ atmosphere. LC-MS showed compound 2 was completelyconsumed and one main peak with desired m/z (calculated MW: 4551.32,observed m/z: 1517.7 ([M/3+H]⁺) and 1138.6 ([M/4+H]⁺)) was detected. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by prep-HPLC (TFAcondition), and BCY13340 (34.7 mg, 7.62 μmol, 59.59% yield, 89.59%purity) was obtained as a white solid.

Example 15: Synthesis of BCY13342

Procedure for Preparation of BCY12860-PEG5-N₃

A mixture of BCY12860 (28.0 mg, 13.58 μmol, 1.0 eq.) and compound 2 (6.5mg, 14.94 μmol, 1.1 eq.) was dissolved in MeCN/H₂O (1:1, 1 mL), and thenthe pH of this solution was adjusted to 8 by dropwise addition of NaHCO₃(0.1 M). The reaction mixture was stirred at 25° C. for 1 hr. LC-MSshowed BCY12860 was consumed completely and one main peak with desiredm/z (MW: 2378.78, observed m/z: 1190.2 ([(M/2+H⁺])) was detected. Thereaction mixture was concentrated under reduced pressure to removesolvent and produced a residue. The residue was then purified byprep-HPLC (neutral condition). BCY12860-PEG5-N₃ (20.7 mg, 8.41 μmol,61.95% yield, 96.7% purity) was obtained as a white solid.

Procedure for Preparation of BCY13342

A mixture of compound 3 (20.7 mg, 8.70 μmol, 1.0 eq.), compound 4 (19.0mg, 9.14 μmol, 1.05 eq.), and THPTA (5.0 mg, 11.31 μmol, 1.3 eq.) wasdissolved in t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂ for3 times), and then CuSO₄ (0.4 M, 28.3 μL, 1.3 eq.) and VcNa (4.5 mg,22.62 μmol, 2.6 eq.) were added under N₂. The pH of this solution wasadjusted to 8 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O),and the solution turned to light yellow. The reaction mixture wasstirred at 25° C. for 2 hr under N₂ atmosphere. LC-MS showed compound 3was consumed completely and one main peak with desired m/z (calculatedMW: 4468.24, observed m/z: 1118.6 ([M/4+H]⁺)) was detected. The reactionmixture was filtered and concentrated under reduced pressure to give aresidue. The crude product was purified by prep-HPLC (TFA condition),and BCY13342 (21.7 mg, 4.60 μmol, 52.85% yield, 94.7% purity) wasobtained as a white solid.

Analytical Data The following heterotandem bicyclic peptide complexes ofthe invention were analysed using mass spectrometry and HPLC. HPLC setupwas as follows:

-   -   Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA in ACN    -   Flow: 1.0 ml/min    -   Column: Gemini-NX C18 5 um 110 A 150*4.6 mm    -   Instrument: Agilent 1200 HPLC-BE(1-614)

Gradients used are described in the table below:

Analytical Method Gradient Description A 25-55% B over 20 minutes B40-70% B over 20 minutes C 45-75% B over 20 minutes D 30-60% B over 20minutes E 45-75% B over 20 minutes F 50-80% B over 20 min 50° C. G35-65% B over 20 minutes H 20-50% B over 20 minutesand the data was generated as follows:

HPLC Complex Retention Analytical ID Analytical Data - Mass SpectrometryTime (min) Method BCY12229 calculated MW: 5125.93, observed m/z: 12.039A 1281.50 ([M/4 + H]⁺ BCY12230 calculated MW: 5225.06, observed m/z:12.085 A 1306.62([M/4 + H]⁺), 1045.0([M/5 + H]⁺) BCY12231 calculated MW:5159.01, observed m/z: 10.359 A 1720.76 ([M/3 + H]⁺), 1291.72 ([M/4 +H]⁺) BCY12232 calculated MW: 5168.02, observed m/z: 12.121 A1722.4([M/3 + H]⁺), 1291.4 ([M/4 + H]⁺) BCY12242 calculated MW: 5159.00,observed m/z: 13.173 A 1719.17([M/3 + H]⁺), 1289.84([M/4 + H]⁺) BCY12375calculated MW: 6064.08, observed m/z: 10.493 B 1516.4([M/4 + H]⁺),1212.8([M/5 + H]⁺) BCY12663 calculated MW: 5383.22, observed m/z: 10.346A 1795.0([M/3 + H]⁺), 1346.7 ([M/4 + H]⁺) BCY12796 calculated MW:5360.18, observed m/z: 11.529 A 1787.7([M/3 + H]⁺), 1341.0([M/4 + H]⁺)BCY12021 calculated MW: 6102.17, observedm/z: 6.822 C 1525.17([M/4 +H]+), 1221.3([M/5 + H]+) BCY12233 calculated MW: 4935.71, observed m/z:10.834 A 1234.16 ([M/4 + H]⁺) BCY12234 calculated MW: 5105.92, observedm/z: 10.58 A 1276.87([M/4 + H]⁺), 1021.80([M/5 + H]⁺) BCY12235calculated MW: 4968.78, observed m/z: 12.165 A 1655.4([M/3 + H]⁺),1242.83([M/4 + H]⁺) BCY12236 calculated MW: 4992.76, observed m/z:10.023 A 1248.4([M/4 + H]⁺) BCY12237 calculated MW: 4992.76, observedm/z: 10.091 A 1248.70([M/4 + H]⁺), 998.70([M/5 + H]⁺) BCY12711calculated MW: 4896.6, observed m/z: 11.23 A 1633.1 ([M/3 + H]⁺),1225.2([M/4 + H]⁺) BCY12712 calculated MW: 4864.63, observed m/z: 11.006A 1622.6([M/3 + H]⁺), 1217.6([M/4 + H]⁺) BCY12713 calculated MW:4863.65, observed m/z: 10.851 A 1621.8([M/3 + H]⁺), 1217.0([M/4 + H]⁺)BCY12714 calculated MW: 4878.67, observed m/z: 10.824 A 1626.9([M/3 +H]⁺), 1220.5([M/4 + H]⁺) BCY12715 calculated MW: 4892.70, observed m/z:10.859 A 1631.6([M/3 + H]⁺), 1223.7([M/4 + H]⁺) BCY12717 calculated MW:4821.61, observed m/z: 11.37 A 1607.8([M/3 + H]⁺), 1206.0([M/4 + H]⁺)BCY12718 calculated MW: 4807.58, observed m/z: 11.527 A 1603.5([M/3 +H]⁺), 1202.9([M/4 + H]⁺) BCY12719 Calculated MW: 4764.57, observed m/z:15.353 H 1192.0 [M/4 + H]⁺, 1589.0 [M/3 + H]⁺ BCY12720 Calculated MW:5319.25, observed m/z: 9.285 B 1065.1 [M/5 + H]⁺, 1331.0 [M/4 + H]⁺,1773.9 [M/3 + H]⁺ BCY12961 Calculated MW: 5105.93, observed m/z: 10.958A 1022.0 [M/5 + H]+, 1277.5 [M/4 + H]+, 1703.0 [M/3 + H]+ BCY12962Calculated MW: 4968.79, observed m/z: 12.517 A 994.9 [M/5 + H]⁺, 1243.3[M/4 + H]⁺, 1656.7 [M/3 + H]⁺ BCY12963 Calculated MW: 4992.76, observedm/z: 10.437 A 1249.0 [M/4 + H]⁺, 1664.9 [M/3 + H]⁺) BCY12964 CalculatedMW: 4992.78, observed m/z: 10.445 A 999.6 [M/5 + H]⁺, 1249.3 [M/4 + H]⁺,1665.3 [M/3 + H]⁺ BCY12965 Calculated MW: 4949.75, observed m/z: 11.302A 990.6 [M/5 + H]⁺, 1238.1 [M/4 + H]⁺, 1650.4 [M/3 + H]⁺ BCY12966Calculated MW: 4963.78, observed m/z: 11.138 A 993.8 [M/5 + H]⁺, 1241.7[M/4 + H]⁺ BCY13029 calculated MW: 4927.69, observed m/z: 11.74 A 1643.7([M/3 + H]⁺), 1233.1 ([M/4 + H]⁺) BCY13030 calculated MW: 4866.62,observed m/z: 11.393 A 1622.7 ([M/3 + H]⁺), 1217.2 ([M/4 + H]⁺), 974.0([M/5 + H]⁺) BCY13031 calculated MW: 4858.59, observed m/z: 10.381 D1619.9 ([M/3 + H]⁺), 1215.1 ([M/4 + H]⁺) BCY13032 calculated MW:4929.67, observed m/z: 13.144 A 1643.9([M/3 + H]⁺), 1233.3([M/4 + H]⁺)BCY13033 calculated MW: 4814.57, observed m/z: 13.497 A 1606.0([M/3 +H]⁺), 1204.2 ([M/4 + H]⁺), 963.6 ([M/5 + H]⁺) BCY13034 calculated MW:4952.75, observed m/z: 12.207 G 1651.5([M/3 + H]⁺), 1239.0([M/4 + H]⁺)BCY13035 calculated MW: 4596.37, observed m/z: 8.083 C 1532.9([M/3 +H]⁺), 1149.9 ([M/4 + H]⁺)) BCY13036 calculated MW: 4573.29, observedm/z: 12.387 D 1525.4([M/3 + H]⁺) and 1143.9([M/4 + H]⁺) BCY13037calculated MW: 4947.74, observed m/z: 11.699 A 1650.4([M/3 + H]⁺),1238.0 ([M/4 + H]⁺) BCY13038 calculated MW: 4870.60, observed m/z:10.747 D 1624.2([M/3 + H]⁺), 1218.5 ([M/4 + H]⁺) BCY13039 calculated MW:4914.67, observed m/z: 10.542 D 1648.5([M/3 + H]⁺), 1235.9 ([M/4 + H]⁺)BCY13040 calculated MW: 4679.45, observed m/z: 14.688 G 1560.8 ([M/3 +H]⁺), 1170.9 ([M/4 + H]⁺), 936.6 ([M/5 + H]⁺) BCY13041 calculated MW:5021.86, observed m/z: 9.609 G 1674.4 ([M/3 + H]⁺), 1256.1 ([M/4 + H]⁺),1005.2 ([M/5 + H]⁺ BCY11616 calculated MW: 4827.46, observed m/z: 12.538A 1609.7([M/3 + H]⁺), 1207.5([M/4 + H]⁺) BCY12238 calculated MW:4763.50, observed m/z: 14.249 A 1587.84 ([M/3 + H]⁺), 1191.37([M/4 +H]⁺) BCY12377 calculated MW: 4668.32, observed m/z: 14.304 A 1556.1([M/3 + H]⁺), 1167.0([M/4 + H]⁺) BCY12379 calculated MW: 4636.32,observed m/z: 10.779 D 1159.8([M/4 + H]⁺) BCY12572 calculated MW:4593.29, observed m/z: 11.303 D 1531.7([M/3 + H]⁺), 1148.8([M/4 + H]⁺)BCY12573 calculated MW: 4579.27, observed m/z: 14.454 A 1526.8([M/3 +H]⁺) BCY12574 calculated MW: 4536.24, observed m/z: 11.959 D1512.9([M/3 + H]+) BCY12575 calculated MW: 5090.92, observed m/z: 9.868C 1698.3([M/3 + H]+), 1273.9([M/4 + H]+) BCY12576 (calculated MW:4705.47, observed m/z: 11.383 D 1568.7 ([M/3 + H]+), 1177.3([M/4 + H]+)BCY12577 Calculated MW: 4719.51, observed m/z: 10.953 D 1180.8 [M/4 +H]+, 1574.2 [M/3 + H]+) BCY12578 Calculated MW: 4662.45, observed m/z:11.906 D 1165.9 [M/4 + H]+, 1554.8 [M/3 + H]+ BCY12579 calculated MW:4784.57, observed m/z: 12.811 A 1595.7([M/3 + H]+), 1197.0([M/4 + H]+)BCY12580 calculated MW: 4776.56, observed m/z: 14.107 A 1592.2 ([M/3 +H]+) BCY12581 calculated MW: 4721.43, observed m/z: 10.776 D 1574.2([M/3 + H]+) and 1181.1 ([M/4 + H]+) BCY12582 calculated MW: 4663.39,observed m/z: 11.002 D 1555.5 ([M/3 + H]+)) BCY12583 calculated MW:4703.38, observed m/z: 11.017 D 1568.6 ([M/3 + H]+), 1176.2([M/4 + H]+)BCY12584 Calculated MW: 5217.13, observed m/z: 12.954 B 1305.1 [M/4 +H]+, 1739.8 [M/3 + H]+) BCY12585 calculated MW: 4735.45, observed m/z:14.077 A 1578.8 ([M/3 + H]+ BCY12709 Calculated MW: 4624.32, observedm/z: 10.749 D 925.5 [M/5 + H]+, 1156.7 [M/4 + H]+, 1541.8 [M/3 + H]+)BCY12710 Calculated MW: 4624.32, observed m/z: 13.832 A 925.6 [M/5 +H]+, 1157.3 [M/4 + H]+, 1541.9 [M/3 + H]+ BCY11468 calculated MW:6783.93, observed m/z: 10.29 B 1131.7([M/6 + H]+ BCY11618 calculated MW:5029.97, observed m/z: 8.89 E 1257.8([M/4 + H]+) and 1006.6([M/5 + H]+BCY11776 calculated MW: 5031.91, observed m/z: 12.598 E 1258.52([M/4 +H]+) and 1006.7([M/5 + H]+) BCY11860 m/z (MW: 5617.56, observed m/z:10.166 F 1404.56 ([(M/4 + H+]) BCY12020 calculated MW: 5709.68, observedm/z: 14.108 B 1902.80([M/3 + H]+), 1427.56([M/4 + H]+ BCY12661calculated MW: 5374.21, observed m/z: 12.408 B 1344.5 ([M/4 + H]+)BCY12969 Calculated MW: 5176.04, observed m/z: 13.147 B 1035.7 [M/5 +H]+, 1294.9 [M/4 + H]+, 1726.8 [M/3 + H]+)

Biological Data

1. CD137 Reporter Assay Co-Culture with Tumour Cells

Culture medium, referred to as R1 media, is prepared by adding 1% FBS toRPMI-1640 (component of Promega kit CS196005). Serial dilutions of testarticles in R1 are prepared in a sterile 96 well-plate. Add 25 μL perwell of test articles or R1 (as a background control) to designatedwells in a white cell culture plate. Tumour cells* are harvested andresuspended at a concentration of 400,000 cells/mL in R1 media. Twentyfive (25) μL/well of tumour cells are added to the white cell cultureplate. Jurkat cells (Promega kit CS196005, 0.5 mL) are thawed in thewater bath and then added to 5 ml pre-warmed R1 media. Twenty five (25)μL/well of Jurkat cells are then added to the white cell culture plate.Incubate the cells and test articles for 6 h at 37° C., 5% CO₂. At theend of 6 h, add 75 μL/well Bio-Glo™ reagent (Promega) and incubate for10 min before reading luminescence in a plate reader (Clariostar, BMG).The fold change relative to cells alone (Jurkat cells+Cell line used inco-culture) is calculated and plotted in GraphPad Prism as log(agonist)vs response to determine EC50 (nM) and Fold Induction over background(Max).

The tumour cell type used in co-culture is NCI-H292 which has been shownto express Nectin-4. The tumour cell type used in co-culture for EphA2is PC3. The tumour cell type used in co-culture for PD-L1 is RKO.

A summary of the fold induction induced by Nectin-4/CD137 heterotandempeptides in the CD137 reporter coculture assay with NCI-H292 cells isshown in Table 1. All compounds are compared to plate control BCY10000which has an average EC50 of 1.1±0.5 nM and Emax of 28±11 fold overbackground.

TABLE 1 Fold induction induced by Nectin-4/CD137 heterotandem bicyclicpeptide complexes in a CD137 reporter assay Fold improvement in Foldimprovement in EC50 over BCY10000 Emax over BCY10000 Complex ID on sameplate on same plate BCY11616 0.21 1.22 BCY12377 0.78 1.37 BCY12379 0.911.29 BCY12572 0.35 1.67 BCY12573 0.74 1.34 BCY12574 0.22 0.77 BCY125750.73 0.35 BCY12576 0.23 1.45 BCY12577 0.25 1.06 BCY12578 0.05 0.93BCY12579 0.29 1.07 BCY12580 0.64 0.97 BCY12581 1.35 0.88 BCY12582 1.691.32 BCY12583 0.60 1.47 BCY12585 1.31 1.50 BCY12709 0.08 2.15 BCY127100.16 1.41 BCY11468 0.45 0.68 BCY11618 0.09 2.59 BCY11776 0.05 0.07BCY11860 0.29 1.53 BCY12020 0.15 0.48 BCY12661 0.34 0.50

A summary of the fold induction induced by EphA2/CD137 heterotandempeptides in the CD137 reporter coculture assay with PC3 cells is shownin Table 2. All compounds are compared to plate control BCY9173 whichhas an average EC50 of 0.54 nM and Emax of 42 fold over background.

TABLE 2 Fold induction induced by EphA2/CD137 heterotandem bicyclicpeptide complexes in a CD137 reporter assay Fold improvement in Foldimprovement in EC50 over BCY9173 Emax over BCY9173 Complex ID Cell lineon same plate on same plate BCY12233 PC3 1.0 0.9 BCY12234 PC3 1.0 0.8BCY12235 PC3 1.1 0.8 BCY12236 PC3 1.4 0.8 BCY12237 PC3 1.0 0.8

A summary of the fold induction induced by PD-L1/CD137 heterotandempeptides in the CD137 reporter coculture assay with RKO cells is shownin Table 3.

TABLE 3 Fold induction induced by PD-L1/CD137 heterotandem bicyclicpeptide complexes in a CD137 reporter assay Fold Induction Bicycle IDEC50(nM) over background BCY12229 18 8 BCY12230 46 10 BCY12242 20 13BCY12375 22 3

2. Pharmacokinetics of CD137 Heterotandem Bicyclic Peptide Complexes inSD Rats

Male SD Rats were dosed with 2 mg/kg of each heterotandem Bicyclepeptide complex formulated in 25 mM Histidine HCl, 10% sucrose pH 7.Serial bleeding (about 80 μL blood/time point) was performed viasubmadibular or saphenous vein at each time point. All blood sampleswere immediately transferred into prechilled microcentrifuge tubescontaining 2 μL K2-EDTA (0.5M) as anti-coagulant and placed on wet ice.Blood samples were immediately processed for plasma by centrifugation atapproximately 4° C., 3000 g. The precipitant including internal standardwas immediately added into the plasma, mixed well and centrifuged at12,000 rpm, 4° C. for 10 minutes. The supernatant was transferred intopre-labeled polypropylene microcentrifuge tubes, and then quick-frozenover dry ice. The samples were stored at 70° C. or below as needed untilanalysis. 7.5 μL of the supernatant samples were directly injected forLC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode todetermine the concentrations of Bicycle. Plasma concentration versustime data were analyzed by non-compartmental approaches using thePhoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T½, AUC(0-last),AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentrationversus time profile were reported. The pharmacokinetic parameters fromthe experiment are as shown in Table 4:

TABLE 4 Pharmacokinetic Parameters in SD Rats Dosing Dose Clp CompoundRoute (mg/kg) T½(h) Vdss (L/kg) (ml/min/kg) BCY12234 IV Inf 2.0 0.420.95 26 BCY13035 IV Inf 3.0 1.5 0.63 11 BCY13040 IV Inf 3.0 1.6 0.63 10

1. A heterotandem bicyclic peptide complex comprising: (a) a firstpeptide ligand which binds to a component present on a cancer cell;conjugated via a linker to (b) a second peptide ligand which binds to acomponent present on an immune cell; wherein each of said peptideligands comprise a polypeptide comprising at least three reactivegroups, separated by at least two loop sequences, and a molecularscaffold which forms covalent bonds with the reactive groups of thepolypeptide such that at least two polypeptide loops are formed on themolecular scaffold, characterised in that said heterotandem bicyclicpeptide complex comprises the following first and second peptideligands: Heterotandem Complex No. First Peptide Second Peptide BCY12229[Ac]D[HArg]CSAGWLTMCQKLHLCPSHAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 1; BCY11865)ID NO: 67; BCY8928) BCY12230 [Ac]D[HArg]CSKGWLTMCQK(Ac)LHLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 2; BCY11866)ID NO: 67; BCY8928) BCY12231 [Ac]D[HArg]CSAGWLTKCQK(Ac)LHLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 3; BCY11867)ID NO: 67; BCY8928) BCY12232 [Ac]D[HArg]CSAGWLTMCKK(Ac)LHLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 4; BCY11868)ID NO: 67; BCY8928) BCY12242 [Ac]D[HArg]CSAGWLTMCQK(Ac)LKLCPSH (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 5; BCY11869)ID NO: 67; BCY8928) BCY12375 Ac-SDKCSAGWLTMCQK[PYA]LHLCPSH (SEQ ID NO:[Ac]C[tBuAla]EE(dK)PYCFADPY[Nle]C[Dap(PYA)] 6; BCY10861)(SEQ ID NO: 68; BCY12023) BCY12663[Ac]SD[HArg]CSAGWLTMCQ[HArg]LHLCPSHK (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 7; BCY12479)ID NO: 67; BCY8928) BCY12796 [Ac]SD[HArg]CSAGWLTMC[HArg]QLNLCPSHK (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 8; BCY12477)ID NO: 67; BCY8928) BCY12021 Ac-SDKCSAGWLTMCQK[PYA]LHLCPSH (SEQ ID NO:[Ac]C[tBuAla]PE[dK]PYCFADPY[Nle]C[Dap(PYA)] 9; BCY10861)(SEQ ID NO: 69; BCY11144) BCY12233[PYA]A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 10; BCY11813)NO: 70; BCY8920) BCY12234 [Ac]A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CK(PYA)Ac-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 11; BCY11814)NO: 70; BCY8920) BCY12235 [Ac]A[HArg]DC[HyP[LVNPLCLK(PYA)P[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 12; BCY11815)NO: 70; BCY8920) BCY12236 [Ac]A[HArg]DC[HyP]K(PYA)VNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 13; BCY11816)NO: 70; BCY8920) BCY12237 [Ac]A[HArg]DC[HyP]LVNPLCK(PYA)HP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 14; BCY11817)NO: 70; BCY8920) BCY12711 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]EE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 71; BCY12143) BCY12712 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 72; BCY12149) BCY12713 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFANPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 73; BCY12147) BCY12714 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFAEPY[Nle]C (SEQ ID NO: 15; BCY9594)NO: 74; BCY12145) BCY12715 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFA[Aad]PY[Nle]C NO: 15; BCY9594)(SEQ ID NO: 75; BCY12146) BCY12717A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle] NO: 15; BCY9594)[Cysteamine] (SEQ ID NO: 76; BCY12352) BCY12718A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID [3-mercaptopropionicNO: 15; BCY9594) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C(SEQ ID NO: 77; BCY12353) BCY12719A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID [3-mercaptopropionicNO: 15; BCY9594) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle][Cysteamine] (SEQ ID NO: 78; BCY12354) BCY12720A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID Palmitic acid-yGly-yGlu-NO: 15; BCY9594) C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO:79; BCY12360) BCY12961 [Ac]A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CKAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 16; BCY12734)ID NO: 67; BCY8928) BCY12962[Ac]A[HArg]DC[HyP]LVNPLCLKP[dD]W[HArg]C (SEQ Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 17; BCY12735)ID NO: 67; BCY8928) BCY12963[Ac]A[HArg]DC[HyP]KVNPLCLHP[dD]W[HArg]C (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 18; BCY12736)ID NO: 67; BCY8928) BCY12964[Ac]A[HArg]DC[HyP]LVNPLCKHP[dD]W[HArg]C (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 19; BCY12737)ID NO: 67; BCY8928) BCY12965 A[HArg]DC[HyP]LVNPLCLHP[dE]W[HArg]C (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 20; BCY12738)ID NO: 67; BCY8928) BCY12966 A[HArg]EC[HyP]LVNPLCLHP[dE]W[HArg]C (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 21; BCY12739)ID NO: 67; BCY8928) BCY13029 A[HArg]DC[HyP]LVNPLCLEP[dD]W[HArg]C (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 22; BCY12854)ID NO: 67; BCY8928) BCY13030 A[HArg]DC[HyP]LVNPLCLHP[dD]WTC SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 23; BCY12855)ID NO: 67; BCY8928) BCY13031 A[HArg]DC[HyP]LVNPLCLEP[dD]WTC SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 24; BCY12856)ID NO: 67; BCY8928) BCY13032 A[HArg]DC[HyP]LVNPLCLEP[dD]WTC[dA] (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 25; BCY12857)ID NO: 67; BCY8928) BCY13033A[HArg]DC[HyP]LVNPLCLEP[dA]WTC SEQ ID NO: 26; Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY12858)ID NO: 67; BCY8928) BCY13034A[HArg]DC[HyP]LVNPLCL[33DPA]P[dD]WTC (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 27; BCY12859)ID NO: 67; BCY8928) BCY13035C[HyP]LVNPLCL[33DPA]P[dD]WTC (SEQ ID NO: 28;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY12860)ID NO: 67; BCY8928) BCY13036 C[HyP]LVNPLCLEP[dD]WTC[dA] (SEQ ID NO: 29;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY12861)ID NO: 67; BCY8928) BCY13037 A[HArg]DC[HyP][Cba]VNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 30; BCY12862)ID NO: 67; BCY8928) BCY13038 A[HArg]DC[HyP][Cba]VNPLCLEP[dD]WTCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 31; BCY12863)ID NO: 67; BCY8928) BCY13039 [dA][HArg]DC[HyP][Cba]VNPLCLEP[dD]WTC[dA]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 32; BCY12864)ID NO: 67; BCY8928) BCY13040 C[HyP][Cba]VNPLCL[33DPA]P[dD]WTC[dA] (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 33; BCY12865)ID NO: 67; BCY8928) BCY13041 A[HArg]DC[HyP]LVNPLCL[33DPA]P[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 34; BCY12866)ID NO: 67; BCY8928) BCY13141A[HArg]DC[HyP]LVNPLCLEP[dD]WTC SEQ ID NO: 24; [3-mercaptopropionicBCY12856) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ IDNO: 77; BCY12353) BCY13142 A[HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]C (SEQ ID[3-mercaptopropionic NO: 15; BCY9594) acid][tBuAla]EE[dK]PYCFADPY[Nle]C(SEQ ID NO: 80; BCY13137) BCY13143A[HArg]DC[HyP]LVNPLCLEP[dD]WTC SEQ ID NO: 24; [3-mercaptopropionicBCY12856) acid][tBuAla]EE[dK]PYCFADPY[Nle]C (SEQ ID NO: 80; BCY13137)BCY13250 A[HArg]DC[HyP]LVNPLCLHP[d1Nal]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 35; BCY13116)ID NO: 67; BCY8928) BCY13251 A[HArg]DC[HyP]LVNPLCL[1Nal]P[dD]W[HArg]CAc-C [tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 36; BCY13117)(SEQ ID NO: 67; BCY8928) BCY13252A[HArg]DC[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 37; BCY13118)ID NO: 67; BCY8928) BCY13253 C[HyP]LVNPLCL[1Nal]P[dD]WTC (SEQ ID NO: 38;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY13119)ID NO: 67; BCY8928) BCY13254[Ac]C[HyP]LVNPLCL[33DPA]P[dD]WTC[dK] (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 39; BCY13120)ID NO: 67; BCY8928) BCY13255 [NMeAla][HArg]DC[HyP]LVNPLCLHP[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 40; BCY13121)ID NO: 67; BCY8928) BCY13256 [NMeAla][HArg]DC[HyP]LVNPLCLEP[dD]WTCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 41; BCY13122)ID NO: 67; BCY8928) BCY13257 [dA][HArg]DC[HyP][Cba]VNPLCLEP[dA]WTC[dA]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 42; BCY13123)ID NO: 67; BCY8928) BCY13258[d1Nal][HArg]DC[HyP][Cba]VNPLCLEP[dA]WTC[dA]Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 43; BCY13124)ID NO: 67; BCY8928) BCY13260 [dA]EDC[HyP]LVNPLCLEP[dD]WTC SEQ ID NO: 44;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY13126)ID NO: 67; BCY8928) BCY13261 [dA][dA]DC[HyP]LVNPLCLEP[dD]WTCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 45; BCY13127)ID NO: 67; BCY8928) BCY13262 ADC[HyP]LVNPLCLEP[dD]WTC (SEQ ID NO: 46;Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ BCY13128)ID NO: 67; BCY8928) BCY13264 A[HArg]DC[HyP][hGlulVNPLCLHP[dD]W[HArg]C Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 47; BCY13130)ID NO: 67; BCY8928) BCY13265 A[HArg]DC[HyP]LVNPLC[hGlu]HP[dD]W[HArg]C  Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 48; BCY13131)ID NO: 67; BCY8928) BCY13266 A[HArg]DC[HyP]LVNPLCL[hGlu]P[dD]W[HArg]C Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 49; BCY13132)ID NO: 67; BCY8928) BCY13268 A[HArg]DC[HyP]LVNPLCLHP[dNle]W[HArg]C Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 50; BCY13134)ID NO: 67; BCY8928) BCY13269 A[HArg]DC[HyP]LVNPLCL[Nle]P[dD]W[HArg]CAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 51; BCY13135)ID NO: 67; BCY8928) BCY13340 C[HyP][Cba]VNPLCL[33DPA]P[dD]WTC[dA] (SEQ[3-mercaptopropionic ID NO: 33; BCY12865)acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 77; BCY12353)BCY13342 C[HyP]LVNPLCL[33DPA]P[dD]WTC (SEQ ID NO: 28;[3-mercaptopropionic BCY12860)acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO: 77; BCY12353)BCY11616 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:Ac-ACIEE(D-K)(PYA)QYCFADPY(Nle)CA (SEQ ID NO: 52; BCY8116) 81; BCY7744)BCY12238 [Ac]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WC (SEQAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ ID NO: 53; BCY12024)ID NO: 67; BCY8928) BCY12377CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:[Ac]C[tBuAla]EE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID 52; BCY8116)NO: 71; BCY12143) BCY12379 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID 52; BCY8116)NO: 72; BCY12149) BCY12572 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle] 52; BCY8116)[Cysteamine] (SEQ ID NO: 76; BCY12352) BCY12573CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: [3-mercaptopropionic52; BCY8116) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C(SEQ ID NO: 77; BCY12353) BCY12574CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: [3-mercaptopropionic52; BCY8116) acid][tBuAla]PE[dK(PYA)]PYCFADPY[Nle][Cysteamine] (SEQ ID NO: 78; BCY12354) BCY12575CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: Palmitic acid-yGly-yGlu-52; BCY8116) C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C (SEQ ID NO:79; BCY12360) BCY12576 [3-mercaptopropionicAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQacid]P[1Nal][dK]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 67; BCY8928)ID NO: 54; BCY12363) BCY12577 [Ac]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ[Cysteamine] (SEQ ID NO: 55; BCY12364) ID NO: 67; BCY8928) BCY12578[3-mercaptopropionic Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQacid]P[1Nal][dK]CM[HArg]DWSTP[HyP]W ID NO: 67; BCY8928)[Cysteamine] (SEQ ID NO: 56; BCY12365) BCY12579[Ac]CP[1Nal][dK]CM[HArg]HWSTP[HyP]WC (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 57; BCY12366)ID NO: 67; BCY8928) BCY12580[Ac]CP[1Nal][dK]CM[HArg]EWSTP[HyP]WC (SEQ IDAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ NO: 58; BCY12367)ID NO: 67; BCY8928) BCY12581CP[1Nal][dE]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 59; BCY12368)ID NO: 67; BCY8928) BCY12582CP[1Nal][dA]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ 60; BCY12369)ID NO: 67; BCY8928) BCY12583 CP[1Nal][dE]CL[HArg]DWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 61; BCY12370)ID NO: 67; BCY8928) BCY12584 Palmitic acid-yGlu-yGlu-Ac-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQCP[1Nal][dK]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: ID NO: 67; BCY8928)62; BCY12371) BCY12585 CP[1Nal][dE]CM[HArg]EWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 63; BCY12384)ID NO: 67; BCY8928) BCY12709CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFAD[NMeAla]Y[Nle]C 52; BCY8116)(SEQ ID NO: 82; BCY12381) BCY12710CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: [Ac]C[tBuAla]PE[dK(PYA)]PYCFAD[NMeDAla]Y[Nle]C 52; BCY8116)(SEQ ID NO: 83; BCY12382) BCY11468[PYA][B-AlalCP[1Nal][dD]CM[HArg]DWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys(PYA)]PYCFADPY[Nle]CA (SEQ (SEQ ID NO: 64; BCY11016)ID NO: 67; BCY8928) BCY11618[PYA][B-Ala]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 65; BCY11143)NO: 70; BCY8920) BCY11776 CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:[Ac]C[tBuAla]PE[dK]PYCFADPY[Nle]C[Dap(PYA)] 52; BCY8116)(SEQ ID NO: 69; BCY11144) BCY11860[PYA][B-Ala]CP[1Nal][dK]CM[HArg]DWSTP[HyP]WCAc-C[tBuAla]PE[D-Lys]PYCFADPY[Nle]CA (SEQ ID (SEQ ID NO: 65; BCY11143)NO: 70; BCY8920) BCY12020 [PYA][B-Ala]CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC[Ac]C[tBuAla]PE[dK]PYCFADPY[Nle]C[Dap(PYA)] (SEQ ID NO: 64; BCY11016)(SEQ ID NO: 69; BCY11144) BCY12661 [PYA[CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC[Ac]C[tBuAla]EE(dK)PYCFADPY[Nle]C[Dap(PYA)] (SEQ ID NO: 66; BCY11015)(SEQ ID NO: 68; BCY12023) BCY12969CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO:[Ac]C[tBuAla]PE[dK(PYA)]PYCFADPY[Nle]C[1,2- 52; BCY8116)diaminoethane] (SEQ ID NO: 84; BCY12358)

wherein 1Nal represents 1-naphthylalanine, HArg represents homoarginine,HyP represents hydroxyproline, B-Ala represents beta-alanine, PYArepresents 4-pentynoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cbarepresents β-cyclobutylalanine, hGlu represents homoglutamic acid, Nlerepresents norleucine, NMeAla represents N-methyl-alanine, tBuAlarepresents t-butyl-alanine, Aad represents alpha-L-aminoadipic acid, Acrepresents an acetyl group, Dap represents diaminopropionic acid, or apharmaceutically acceptable salt thereof.
 2. The heterotandem bicyclicpeptide complex as defined in claim 1, wherein the immune cell isselected from: white blood cells, lymphocytes (e.g. T lymphocytes or Tcells, B cells or natural killer cells), CD8 cells, CD4 cells, dendriticcells, follicular dendritic cells and granulocytes.
 3. The heterotandembicyclic peptide complex as defined in claim 1, wherein the secondpeptide ligand comprises a CD137 binding bicyclic peptide ligand.
 4. Theheterotandem bicyclic peptide complex as defined in claim 3, wherein theCD137 binding bicyclic peptide is selected from any of the peptides ofSEQ ID NOS: 67 to
 84. 5. The heterotandem bicyclic peptide complex asdefined in claim 1, wherein the first peptide ligand comprises aNectin-4 binding bicyclic peptide ligand.
 6. The heterotandem bicyclicpeptide complex as defined in claim 5, wherein the Nectin-4 bindingbicyclic peptide is selected from any of the peptides of SEQ ID NOS: 52to
 66. 7. The heterotandem bicyclic peptide complex as defined in claim5, which is selected from any one of the complexes listed in Table C,such as BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661 andBCY12969.
 8. The heterotandem bicyclic peptide complex as defined inclaim 1, wherein the first peptide ligand comprises an EphA2 bindingbicyclic peptide ligand.
 9. The heterotandem bicyclic peptide complex asdefined in claim 8, wherein the EphA2 binding bicyclic peptide isselected from any of the peptides of SEQ ID NOS: 10 to
 51. 10. Theheterotandem bicyclic peptide complex as defined in claim 8, which isselected from any one of the complexes listed in Table B, such asBCY13035, BCY13040, BCY13253, BCY13254, BCY13340 and BCY13342.
 11. Theheterotandem bicyclic peptide complex as defined in claim 1, wherein thefirst peptide ligand comprises a PD-L1 binding bicyclic peptide ligand.12. The heterotandem bicyclic peptide complex as defined in claim 11,wherein the PD-L1 binding bicyclic peptide is selected from any of thepeptides of SEQ ID NOS: 1 to
 9. 13. The heterotandem bicyclic peptidecomplex as defined in claim 11, which is selected from any one of thecomplexes listed in Table A, such as BCY12375 and BCY12021.
 14. Theheterotandem bicyclic peptide complex as defined in claim 1, wherein themolecular scaffold is selected from1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).
 15. Theheterotandem bicyclic peptide complex as defined in claim 1, wherein thepharmaceutically acceptable salt is selected from the free acid or thesodium, potassium, calcium, ammonium salt.
 16. A pharmaceuticalcomposition which comprises the heterotandem bicyclic peptide complex ofclaim 1 in combination with one or more pharmaceutically acceptableexcipients.
 17. A method of preventing, suppressing or treating cancer,which comprises administering to a patient in need thereof theheterotandem bicyclic peptide complex of claim 1.